Characterization of Anaphylatoxin Receptor Expression and C3a/C5a Functions in Anaphylatoxin Receptor Reporter Mice

The anaphylatoxins (AT) C3a and C5a are effector molecules of C3 and C5 exerting multiple biologic functions through binding and activation of their cognate G protein−coupled receptors. C3a interacts with the C3a receptor (C3aR), whereas C5a and its primary degradation product C5a‐desArg engage C5aR1 and C5aR2. In the past, analysis of AT expression has been hampered by cross reaction of antibodies designed to recognize the different AT receptors. Furthermore, assessment of effects mediated by cell‐specific activation has been difficult. Here, floxed AT receptor reporter mice are described as tools to monitor AT receptor expression in cells and tissues and to study the functions of C3a and C5a by cell‐specific deletion of their cognate AT receptors. © 2020 The Authors.


INTRODUCTION
In recent years, our view of the complement system as a guardian of the extracellular space has been markedly extended. Several reports have shown that complement is not activated systemically solely via the three canonical activation pathways, i.e., the classical, lectin, and alternative pathway, but by non-canonical pathways via specific proteases derived from pathogens, activated host cells, or the contact system (Hajishengallis, Reis, Mastellos, Ricklin, & Lambris, 2017). These newly discovered non-canonical activation pathways of complement, its unexpected contributions to cell homeostasis, metabolism, differentiation, and apoptosis, its crosstalk with several receptor classes and other cascade systems of innate immunity, as well as its impact on adaptive immune responses make the complement system more than ever an important field of research within immunology (Kolev, Le Friec, & Kemper, 2014;West, Kolev, & Kemper, 2018).
Many of these newly discovered functions can be attributed to the anaphylatoxins (ATs) C3a and C5a, which were originally considered mere proinflammatory molecules. Their pleiotropic functions are mainly mediated through activation of their G protein−coupled corresponding complement peptide receptors, i.e., C3aR for C3a and C5aR1 and C5aR2 for C5a. Until recently, AT receptor expression in professional and non-professional immune cells, as well as tissue-resident stroma cells, has been ill-defined, and is still controversial (Laumonnier, Karsten, & Köhl, 2017). To better understand the expression patterns of the AT receptors in health and disease, and to define the multiple roles of the ATs in the innate and adaptive immune networks, we have generated floxed AT receptor reporter mice that are now available to and widely used by the scientific community.
This article describes the use of C3aR and C5aR2 reporter mice with a tandem-dye (td)Tomato construct Quell et al., 2017), as well as the use of a C5aR1 knock-in mouse in which the Aequorea coerulescens green fluorescent protein [(Ac)GFP] was added to monitor C5aR1 expression (Karsten et al., 2015). These mice are available from the Köhl laboratory upon request. Basic Protocol 1 outlines the procedure to genotype GFP-C5aR1 knockin mice. Support Protocol 1 describes as an example the genotyping of mice with conditional deletion of C5aR1 in LysM-expressing cells after Cre-mediated targeting. Basic Protocol 2 describes the genotyping of tdTomato-C3aR and C5aR2 knock-in mice, with Support Protocol 2 detailing the preparation of the genomic DNA. Basic Protocol 3, together with two support protocols that describe the analysis of AT receptor protein (Support Protocol 3) and mRNA (Support Protocol 4) expression, outlines a flow cytometric approach to assess AT receptor expression in GFP-C5aR1 + , tdTomato-C5aR2 + , or tdTomato-C3aR + cells. To link the expression of the AT receptor reporter constructs with AT receptor function, we provide three basic protocols to determine distinct functions of C5aR1 in GFP-C5aR1 knockin mice (Basic Protocol 4), C3aR in tdTomato-C3aR knockin mice (Basic Protocol 5), and C5aR2 in tdTomato-C5aR2 knockin mice (Basic Protocol 6). Finally, we have added Support Protocol 5 for the isolation of NK cells, which exclusively express C5aR2 but not C5aR1.
Landwirtschaft, Umwelt und Ländliche Räume, Kiel, Germany). All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

GENOTYPING OF FLOXED GFP-C5aR1 KNOCKIN MICE
The genes encoding for the three AT or complement peptide receptors are organized in a similar way, with a 5 -untranslated region (UTR), two exons, and a 3 -UTR after exon 2 (https:// www.guidetopharmacology.org/ GRAC/ FamilyDisplayForward?familyId=5). This offers the opportunity to insert a fluorescent reporter sequence (tdTomato or GFP) before or at the beginning of exon 2. With the inserted construct, one locus of cross-over P1 (loxP) site is introduced at the 5 end of the reporter sequence and another one after the coding region of the receptor before the 3 -UTR. According to the general design, the C5aR1 construct includes a GFP coding sequence ending with a stop codon, followed by an internal ribosomal entry site (IRES) placed upstream of exon 2 encoding for the C5ar1 gene, which is flanked by loxP sites at both ends. Between the loxP site at the 5 -UTR  Abbreviations: bp, base pair; F, forward; GFP, green fluorescent protein; Il2, IL-2 precursor short; R, reverse; Tcrd, T cell receptor delta chain. and the GFP reporter gene, a neomycin cassette with flippase recognition target (FRT) sites at both ends was included. During the generation of mice, the neomycin cassette was removed by breeding to a flippase (FLP) deleter mouse strain ( Fig. 1).
To determine the genotype of the AT receptor reporter mice, we have set up a PCR-based approach in which we detect the presence of the reporter sequence (GFP: PCR1) including the loxP sites and/or the AT receptor wild-type genes (C5ar1: PCR 2), in particular in the case of target cell−specific AT receptor deletion after breeding of the AT receptor reporter mice to defined Cre mice (H. Kim, Kim, Im, & Fang, 2018;Song & Palmiter, 2018). In the latter case, the Cre mouse strain used needs to be genotyped in a separate approach, as outlined in Support Protocol 1.

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Current Protocols in Immunology Additional reagents and equipment for agarose gel electrophoresis (Brody & Kern, 2004; also see Current Protocols article Voytas, 2001) CAUTION: Remember the proper work safety requirements to protect eyes and skin against UV radiation. Boric acid belongs to the group of carcinogenic, mutagenic, reprotoxic (CMR) substances. Follow the national requirements for disposal and working safety.
NOTE: All chemicals need to be molecular grade (DNase-free). For all steps use filtered pipet tips only.
1. Prepare PCR1 primer mix by combining 20 μl each of primers AH7/AH9/GK91/ GK92 (Table 1)  4. Combine 5 μl template [genomic DNA (Support Protocol 2) from floxed GFP-C5aR1 knockin mouse diluted 1:10 with nuclease-free water] or nuclease-free water for control with 20 μl of the appropriate master mix in a 0.2-ml 8-tube PCR strip without caps, and run the following thermocycling conditions for both PCR1 and PCR2 after closing the tubes with the 8-capstrips for PCR tubes: Step no.

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Current Protocols in Immunology workstation (e.g., Analytic Jena) to avoid cross contamination and a PCR cooler (e.g., Eppendorf AG) to secure the template quality.
Also see Current Protocols article: Voytas (2001  The primer combination AH7/AH8 amplifies a 609-bp DNA fragment of the C5ar1 gene (from exon 2) in wild-type and GFP-C5aR1 fl/+ mice. In GFP-C5aR1 fl/+ and GFP-C5aR1 fl/fl mice, it amplifies an additional 2037-bp DNA fragment comprising the GFP gene, the IRES fragment, and a part of exon 2 of the C5ar1 gene. The primer combination AH15/AH16 amplifies a 324-bp DNA fragment of the IL-2 precursor gene as an internal template control in samples from all strains. Amplification products were separated on a 2.5% (PCR1) or 1.5% (PCR2) sodium borate (SB) agarose gel stained with GelRed. M = marker; W = water control; 1 = wild type; 2 = heterozygous GFP-C5ar1 fl/+ mice; 3 = homozygous GFP-C5ar1 fl/fl mice. Tilting on a conventional rocking platform with ±7.5°at about 15 rpm will ensure uniform staining results. No destaining step is necessary. GelRed is non-mutagenic and safer for the environment than ethidium bromide.

Visualize the gel bands with UV light and document the result by photography.
Typical results showing the amplicons from genotyping of wild-type, GFP-C5aR1 fl/+ , and GFP-C5aR1 fl/fl mice are shown in Figure 2. The sizes of the PCR fragments amplified from the different mouse strains are shown in Table 2.

GENOTYPING OF LysMcre-C5aR1 MICE
Breeding of the floxed GFP-C5aR1 knockin mice with strains that harbor a Cre recombinase will result in the targeted deletion of the GFP-C5aR1 cassette through excision at the two loxP sites (Fig. 1D). Similarly, the tdTomato-C3ar1 or C5ar2 genes will be excised in response to Cre (Figs. 4D and 5D). Here, we describe the genotyping of LysMcre-C5aR1 in which LysMcre mice (Clausen, Burkhardt, Reith, Renkawitz, & Forster, 1999) have been crossed with floxed GFP-C5aR1 knockin mice to specifically delete C5aR1 in granulocytes, monocytes, and macrophages (Karsten et al., 2015). Given that the C5ar1 gene is still expressed in many cell types, we have set up an analysis system that includes C5ar1 (Basic Protocol 1) and Lyz2, the gene encoding for lysozyme M(lysM). The C5ar1 and Lyz2 genes are expressed on two different chromosomes, i.e., chromosomes 7 and 10. To delete C5aR1 in lysM-expressing cells, GFP-C5aR1 fl/fl mice need to harbor at least one functional Cre allele under the control of the lysM promoter.  Sarstedt,cat. no. 72.690) PCR machine (BioRad C1000 or C1000 Touch Thermal Cycler) Gel staining tray UV transilluminator and camera Additional reagents and equipment for agarose gel electrophoresis (Brody & Kern, 2004; also see Current Protocols article Voytas, 2001) CAUTION: Remember the proper work safety requirements to protect eyes and skin against UV radiation. Boric acid belongs to the group of carcinogenic, mutagenic, reprotoxic (CMR) substances. Follow the national requirements for disposal and working safety.

Materials
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Current Protocols in Immunology  Figure 3. The sizes of the PCR fragments amplified from the different mouse strains are shown in Table 4.

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Current Protocols in Immunology

GENOTYPING OF FLOXED tdTomato-C3aR AND tdTomato-C5aR2 KNOCKIN MICE
In addition to C5aR1, we have targeted the C3ar1 and C5ar2 genes to generate reporter mice that allow tracking of cellular C3aR and C5aR2 expression and cell-specific deletion. To provide a strong signal with a high stability, in particular upon standard fixation procedures, we used a tandem-dye (td)Tomato instead of GFP (de Felipe et al., 2006). Furthermore, although IRES is a widely used design to produce independent proteins from one mRNA, we and others have found that the translation of the coding sequence under the control of IRES is less efficient than the translation of the main ribosomal entry sequence (de Felipe et al., 2006;Karsten et al., 2015). As an alternative approach, we have used the short, self-cleaving peptide derived from porcine teschovirus-1 (P2A; Kim et al., 2011), which shows high cleavage efficiency and a stoichiometric expression of proteins flanking the 2A peptide (de Felipe et al., 2006;Kim et al., 2011). In the case of the C3ar1 gene, the resulting targeting construct encoded a 6.2-kb 5 homology arm and a 3.0-kb 3 homology arm flanking the floxed coding region of exon 2, in which tdTomato_P2A has been inserted in frame with the ATG of exon 2 (Fig. 4). Similarly, we have inserted the tdTomato sequence following the splice acceptor of exon 2 in-frame with the coding sequence of the C5ar2 gene (Fig. 5). In both cases, two loxP sites flank the constructs for potential excision of the C3ar1 or C5ar2 genes by Cre-mediated deletion.

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Gel staining tray UV transilluminator and camera
Additional reagents and equipment for agarose gel electrophoresis (Brody & Kern, 2004; also see Current Protocols article Voytas, 2001) CAUTION: Remember the proper work safety requirements to protect eyes and skin against UV radiation. Boric acid belongs to the group of carcinogenic, mutagenic, reprotoxic (CMR) substances. Follow the national requirements for disposal and working safety.
NOTE: All chemicals need to be molecular grade (DNase free). For all steps use filtered pipet tips only.
You can store the PCR primer mixes at −20°C for several months before use.
4. Prepare master mixes for PCR 4, 5, and 6 separately by pipetting the following reagents at the indicated volumes into a 1.5-ml microcentrifuge tube (per sample): 6.25 μl nuclease-free water 1.25 μl primer mix (PCR4, 5 or 6) 12.5 μl 2× KAPA2G Fast Hot Start Genotyping Mix. Mix and microcentrifuge briefly to bring mixture to bottom of tube.
5. Combine 5 μl template [genomic DNA (Support Protocol 2) from floxed tdTomato-C3aR knockin mouse or -tdTomato-C5aR2 knockin mouse diluted 1:10 with nuclease-free water] or nuclease-free water for control with 20 μl of the respective master mix in a 0.2-ml 8-tube PCR strip without caps and run the following thermocycling conditions for PCR4 after closing the tubes with the 8-capstrips for PCR tubes: Step no.

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Current Protocols in Immunology  6. Run samples on a 2.5% (PCR5 and 6) or an 1% (PCR4) SB agarose gel (Brody & Kern, 2004; also see Current Protocols article Voytas, 2001 Typical results showing the amplicons of wild-type, tdTomato-C3aR1 fl/+ and tdTomato-C3aR1 fl/fl mice are depicted in Figure 6. The size of the PCR fragments amplified from the different mouse strains are shown in Table 6.
Typical results showing the amplicons of wild-type, tdTomato-C5aR2 fl/+ and tdToamto-C5aR2 fl/fl mice are depicted in Figure 7. The size of the PCR fragments amplified from the different mouse strains are shown in Table 7.

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PREPARATION OF GENOMIC DNA
The prerequisite to amplify the different genomic regions of interest by the PCRs outlined in Basic Protocols 1 and 2 is the appropriate preparation of genomic DNA extracted from a piece of tissue. The most frequently used mouse tissues for genomic DNA preparation are from tail or ear. Our animal facility uses ear punch as the method of identification. Thus, we are using such ear punch biopsies for preparation of genomic DNA as outlined below. Several methods and kits are available to extract DNA from mouse tissue. We routinely use the KAPA Express Extract Kit, which results in high amounts of genomic DNA of good quality.
1. Transfer biopsy using a Blunt Fill cannula from the tube that was used to collect the specimen from the animal into a 0.2-ml PCR tube with lid under sterile conditions.
Make sure that the biopsy is immediately stored at −20°C. In our hands, storing the biopsy for several months does not affect the yield of genomic DNA. Due to static charging, the tissue sticks to the metal during the transfer into the PCR tube.
2. Add 50 μl lysis master mix to the biopsy, vortex, and spin down in a microcentrifuge at room temperature.
The tissue needs to be completely covered by the buffer. Any included air will lead to insufficient lysis, and should be removed during the centrifugation. If necessary, you may press the biopsy down with a pipet tip. In our hands, it is possible to handle up to 32 biopsies simultaneously.
3. Incubate 10 min at 75°C followed by 5 min at 95°C using a cycler or two independent heating blocks.
Avoid opening of the microcentrifuge tubes, due to vapor pressure, which may result in a loss of material and or cross contamination of your sample.
This will pellet cellular debris.
Samples can be stored for years −20°without quality loss. However, avoid repeated thawing and freezing cycles.

DETERMINATION OF C5aR1, C5aR2, AND C3aR EXPRESSION USING FLOXED AT RECEPTOR REPORTER MICE
Floxed AT receptor reporter mice allow the assessment of AT receptor expression without the need for labeled ligands or antibodies. The GFP and tdTomato reporter genes that are under the control of the promoters of the C5ar1, C5ar2, or C3ar1 genes encode for strongly fluorescent proteins that can easily be detected by imaging technologies, including flow cytometry and fluorescence microscopy. As an example, we describe here a protocol to monitor the expression of tdTomato as a surrogate for C3aR expression in peritoneal macrophages. The same procedure can be used to delineate the expression of GFP as a surrogate for C5aR1 and tdTomato as a surrogate for C5aR2 expression, since peritoneal macrophages are known to strongly express all AT receptors (Karsten et al., 2015;Karsten et al., 2017;Quell et al., 2017). Importantly, the genetic association of a reporter gene to AT receptor genes can impact the trafficking of the AT receptors Laumonnier et al.
to the cell surface (Dunkelberger, Zhou, Miwa, & Song, 2012). Thus, it is important to also assess the surface expression of ATRs in GFP-C5aR1 + , tdTomato-C5aR2 + , or tdTomato-C3aR + cells using AT-receptor-specific antibodies, and control for the specificity using the respective AT receptor−deficient mice. For this purpose, we provide Support Protocol 3, which determines C3aR expression using a C3aR-specific antibody. Finally, Support Protocol 4 outlines the assessment of AT receptor mRNA expression in GFP-C5aR1 + , tdTomato-C5aR2 + , or tdTomato-C3aR + cells to verify the AT receptor expression using GFP-C5aR1 mice as an example.
Depending on the background of the AT receptor reporter mice, C57BL/6 or BALB/c mice can serve as control mice to determine the baseline fluorescence for the GFP or the tdTomato signal (see Fig. 15). We regularly use 8-to 12-week-old mice of both sexes. While one mouse of each strain is sufficient to determine AT receptor expression, at least three to five animals should be used to confirm expression and to compare expression in different cell types.

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Current Protocols in Immunology The anti-CD16/32 antibody binds to Fcγ RII and Fcγ RIII, thus preventing non-specific binding to lineage-specific antibodies that define the peritoneal macrophages in peritoneal lavage cells.
6. Discard the supernatant by aspiration.
7. Break up the pellet by gently tapping the bottom of the tube.
8. Prepare an antibody master mix by diluting the anti-F4/80 antibody to a final concentration of 0.5 μg/ml and the anti-CD11b antibody to 0.25 μg/ml in 100 μl D-PBS/BSA. 9. Incubate the cells from each sample with 100 μl of the antibody master mix for 20 min at 4°C.
11. Discard the supernatant by careful aspiration.
12. Resuspend the cells in 300 μl D-PBS/BSA and transfer to a flow cytometer. Figure 8A. Label the population "singlets."

Plot FSC-H versus FSC-W and gate on singlet cells as shown in
The FSC-A should be set at 25,000 to exclude any debris. This will ensure that the events counted will be cells and not any contaminants. Record 5 × 10 5 events for cell analysis.
15. Record the tdTomato signal in the PE channel (also in case of tdTomato-C5aR2 knockin mice) or the GFP-signal in the FITC channel (in case of floxed GFP-C5aR1 knockin mice) (Fig. 8A).
The tdTomato + cells define either the tdTomato-C3aR + or tdTomato-C5aR2 + macrophages, whereas the GFP + cells define the GFP-C5aR1+ cells (depending on the mouse strain used). The autofluorescent emission of the CD11b + F4/80 + macrophages in wild-type mice defines the baseline fluorescence for the GFP or the tdTomato signal (fluorescence minus one (FMO) control).

DETERMINATION OF C3aR EXPRESSION USING A C3aR-SPECIFIC ANTIBODY
The expression of the GFP or tdTomato AT receptor surrogate fluorochrome is not necessarily linked with surface or intracellular expression of the AT receptors. Although our previous results show a strong match of C5aR1 and C3aR surface expression with the GFP or tdTomato signal (Karsten et al., 2015;Quell et al., 2017), we strongly recommend also staining the GFP-C5aR1 + , tdTomato-C3aR + , or tdTomato-C5aR2 + cells with AT receptor−specific antibodies to verify AT receptor surface expression. In the case of tdTomato-C5aR2 + cells, we have not yet been able to verify the surface expression with C5aR2-specific antibodies, as all antibodies that we have tested so far have also showed surface staining in C5aR2-deficient mice, suggesting cross-reactivity with other structures . We strongly recommend using AT receptor−deficient mice to control for the specificity of a given AT receptor antibody and not to rely only on FMO or IgG isotype controls. Support Protocol 3 outlines an example of C3aR staining using an anti-C3aR antibody and an AF647-labeled secondary antibody, as no conjugated C3aR antibodies are commercially available yet. Alternatively, the anti-C3aR antibody might be conjugated with a fluorochrome by commercially available conjugation kits. For C5aR1 staining, several antibodies conjugated with different fluorochromes are available. We routinely anesthetize mice using 300 μl ketamine (100 mg/kg body weight) and xylazine (7.5
We regularly use 8-to 12-week-old mice of both sexes. While one mouse of each strain is sufficient to determine C3aR receptor expression, at least three to five animals should be used to confirm expression and to compare expression in different cell types. 14. Discard the supernatant by aspiration.

Count cells (see Current
15. Break up the pellet by tapping the bottom of the tube.
16. Add 100 μl of the CD11b/F4/80 antibody master mix to each tube and incubate the cells for 20 min at 4°C.
18. Discard the supernatant by aspiration.
19. Resuspend the cells for flow cytometric analysis in 300 μl D-PBS/BSA and analyze the sample with a flow cytometer according to the following steps.
21. Record the tdTomato-C3aR signal in the PE channel and C3aR-AF647 emission in the APC channel (Fig. 8B).

DETERMINATION OF C5aR1, C5aR2, AND C3aR mRNA EXPRESSION IN FLOXED GFP-C5aR1, -tdTomato-C5aR2, OR -tdTomato C3aR−POSITIVE CELLS
In addition to the determination of AT receptor expression using the GFP or tdTomato signal of floxed AT receptor knockin mice or the signal from fluorochrome-labeled AT receptor antibodies, Support Protocol 4 describes an example of the analysis of AT receptor mRNA expression in peritoneal macrophages from floxed GFP-C5aR1 knockin mice. This procedure is useful to verify AT receptor expression when AT receptor antibody staining has revealed cross-reactivity with structures in AT receptor-deficient mice, as is frequently observed with anti-C5aR2 antibodies. We routinely anesthetize mice using 300 μl ketamine (100 mg/kg body weight) and xylazine (7.5 mg/kg body weight) via intraperitoneal injection.
We regularly use 8-to 12-week old mice of both sexes. While one mouse of each strain is sufficient to determine AT receptor expression, at least three to five animals should be used to confirm expression and to compare expression in different cell types. : Strober, 2001). Adjust the cells from each mouse strain to a density of 1 × 10 7 cells/ml in DMEM medium without serum, add 500 μl/well to adherent 24-well-plates, and incubate 3 hr.

Discard the supernatant with the non-adherent cells.
5. Carefully wash the plate with 1 ml D-PBS, to remove remaining non-adherent cells.
6. Add 1 ml D-PBS to each well.
7. Harvest the adherent cells using a cell scraper into 1 ml of D-PBS and transfer detached cells into a 1.5-ml microcentrifuge tube.
8. Wash cells three times, each time with 1 ml D-PBS by centrifuging 5 min at 500 × g, 4°C.
For the following steps, it is important to work quickly and on ice to prevent degradation of the mRNA.

Discard the supernatant and isolate the RNA using the RNeasy Mini Kit according to the manufacturer's instructions.
A minimum of 5 × 10 5 cells should be collected as a cell pellet, but not more than 1 × 10 7 cells 10. Determine the concentration of isolated total RNA using a spectrophotometer (Nan-oDrop or equivalent).
11. Transfer up to 8 μl of total RNA into a fresh autoclaved 1.

5-ml microcentrifuge tube
The total amount of RNA used for the reverse transcription reaction should not exceed 1 μg. In case your RNA yield is >1 μg, adjust the concentration to 1 μg with nuclease free water.
17. Heat the sample for 5 min in a heat block (65°C).

Heating ensures the linearization of the mRNA by breaking off mRNA hairpins prior incubation with the oligo(dT).
18. Place the sample immediately on ice.

Placing the tubes on ice will result in immediate cooling of the solution, preventing the re-formation of secondary structures and favoring the maintenance of linear molecules. This will allow appropriate hybridization of oligo(dT) primers to poly(A) tails of mRNA.
19. Spin down 5 s at 21,000 × g, add 5 μl of 5× RT buffer, and mix by tapping the bottom of the tube.

Remove 5 μl of the reaction and set aside.
This sample will serve as the negative RT control.  28. Add 23 μl of PCR master mix to each of the three tubes.
29. Place the microtube strip into a thermal cycler and run the following thermal cycling conditions: Step no.

Current Protocols in Immunology
Typical results showing the amplicons of C5ar1 from peritoneal macrophages of C57BL/6 wild-type mice and GFP-C5aR1 knockin animals are shown in Figure 9. The amplicon sizes for C5ar2 and C3ar1 are 153 and 101 bp, respectively.

ANALYSIS OF C5aR1-DRIVEN ERK1/2 PHOSPHORYLATION IN GFP-C5aR1 + CELLS
C5aR1 is a G protein−coupled receptor (GPCR) that couples preferentially to pertussis toxin (PT)−sensitive G protein Gai2 or Gai3, but can also bind to the PT-insensitive G proteins Ga15 and Ga16 (Klos, Wende, Wareham, & Monk, 2013). Engagement of C5aR1 by its natural ligands C5a and C5a-desArg results in a series of signaling events that involve the activation of PI3K, Akt, and MAPK signaling, resulting in the phosphorylation of the MAPK ERK 1/2, in particular in bone marrow−derived neutrophils. Thus, phosphorylation of ERK1/2 can be used to assess the C5aR1 function in GFP-C5aR1 + cells. As an example, this protocol describes the analysis of C5a-driven ERK1/2 phosphorylation in GFP-C5aR1 + bone marrow−derived neutrophils. We routinely anesthetize mice using 300 μl ketamine (100 mg/kg body weight) and xylazine (7.5

Isolate bone marrow cells as described in the Current Protocols article
While one mouse of each strain is sufficient to harvest enough bone marrow cells to determine ERK phosphorylation, at least three to five animals should be used to validate the data.

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Current Protocols in Immunology 3. Count cells (see Current Protocols article: Strober, 2001). Adjust the number of bone marrow cells to 1.0 × 10 7 cells/ml using D-PBS.
4. Add 100 μl of the bone marrow cells to a 0.5-ml low-retention tube and let the cells rest for 5 min at 37°C.
Due to their basic pK, C3a and C5a have a very strong capacity to bind plastics. Thus, it is mandatory to use low-retention tubes and tips. 5. Add rhC5a at a final concentration of 10 nM using low-retention pipet tips.
6. Incubate the cells for 5 min at room temperature.
7. Quickly add 4 μl of 37% formaldehyde solution to the 100 μl cell suspension and place cells immediately on ice. 8. Keep the cells for 10 min on ice in the dark.
10. Aspirate the supernatant and break up the pellet by gently tapping the bottom.

Once in methanol, cells can be stored at −20°C for up to 4 weeks.
12. Centrifuge the cells 5 min at 500 × g, 4°C.
The speed of 500 × g must not be exceeded after permeabilization, as cell integrity can be easily destroyed. Also, a washing step is not recommended because of the difficulty in pelleting the cells.

Because the cells do not always form a clear and visible pellet after centrifugation, take extra care while removing the supernatant.
14. Prepare Fc blocking buffer by diluting anti-CD16/32 antibody to a final concentration of 100 μg/ml in D-PBS.
15. Carefully resuspend the cells in 100 μl Fc blocking buffer and incubate the cells for 15 min at 4°C.
18. Prepare an antibody master mix by diluting anti-pERK antibody to a final concentration of 10 μg/ml and anti-Ly6G antibody to 5 μg/ml in D-PBS.
19. Incubate the cells with 100 μl of the antibody master mix for 60 min at 4°C.
Ly6G is a specific marker to define the neutrophil population within the bone marrow cells. Antibodies recognizing  are not recommended, as they also stain monocytes (Daley, Thomay, Connolly, Reichner, & Albina, 2008

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ASSESSMENT OF C3aR FUNCTIONS IN CELLS OBTAINED FROM FLOXED tdTomato-C3aR KNOCKIN MICE
Engagement of C3aR by C3a on different human cell types including neutrophils, eosinophils, and endothelial cells results in activation of PT-sensitive and PT-insensitive G proteins, as well as a transient increase in intracellular Ca 2+ which requires extracellular Ca 2+ sources (Klos et al., 2013). Further, C3aR activation in mouse bone marrow−derived macrophages (Cui, Wu, Song, Chen, & Wan, 2019) has resulted in ERK1/2 phosphorylation. In contrast, we found only minor ERK1/2 phosphorylation in peritoneal macrophages that express high levels of C3aR at their surface (Quell et al., 2017), demonstrating that signaling pathways downstream of C3a do not always result in ERK1/2 phosphorylation. Here, we provide two assays that we have used to determine C3aR function in different cell types, i.e., the internalization of the receptor upon C3a ligation and the assessment of changes in intracellular Ca 2+ concentration [Ca 2+ ] i . These functions can be determined by flow cytometry and fluorescence microscopy.

mg/kg body weight) via intraperitoneal injection.
We regularly use 8-to 12-week-old mice of both sexes.
2. Isolate peritoneal exudate cells from the peritoneum of BALB/c wild-type and tdTomato-C3aR knockin mice as described in the Current Protocols article Zhang et al. (2008).
While one mouse of each strain is sufficient to harvest enough peritoneal cells to determine C3aR internalization, at least three to five animals should be used to validate the data. 7. Add 2 μl of the hC3a working solution to three of the four low-retention 1.5-ml microcentrifuge tubes prepared in step 4 (10 nM final concentration). Add 2 μl D-PBS to one of the four low-retention 1.5-ml microcentrifuge tubes as a control.

Count cells (see Current
8. Gently mix the cells by pipetting up and down and incubate one of three hC3astimulated tubes for 1 min, the second one for 3 min, and the third one for 9 min at 37°C in a heat block. Also, incubate the unstimulated control tube for 9 min at 37°C on the heat block.
9. Add 2 μl of 37% formaldehyde and immediately transfer the tubes on to a pre-cooled rack placed on ice and incubate on ice for 30 min.

The 1.5% formaldehyde solution will stop the reaction and fix the cells.
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Current Protocols in Immunology CAUTION: Formaldehyde belongs to the group of carcinogenic, mutagenic, reprotoxic (CMR) substances. Follow the national requirements for disposal and working safety.
11. Discard the supernatant by careful aspiration.

Resuspend the fixed cells in 100 μl D-PBS/20% FBS and incubate for 30 min at 4°C.
This step will block unspecific binding of the anti-C3aR.
14. Add F4/80-specific antibody to each microcentrifuge to to reach a final concentration of 0.5 μg/ml, and incubate the cells for 20 min at 4°C.
16. Discard the supernatant by aspiration.

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Current Protocols in Immunology 17. Resuspend the cells in each tube in 300 μl of D-PBS/BSA and measure the samples on a flow cytometer. Figure 11A shows the gating strategy to define the F4/80 + peritoneal macrophages and the rapid internalization of the tdTomato-C3aR upon C3a stimulation.

mg/kg body weight) via intraperitoneal injection.
We regularly use 8-to 12-week-old mice of both sexes.
2. Isolate peritoneal exudate cells from the peritoneum of BALB/c wild-type and tdTomato-C3aR knockin mice as described in the Current Protocols article Zhang et al. (2008).
While one mouse of each strain is sufficient to harvest enough peritoneal cells to determine C3aR internalization, at least three to five animals should be used to validate the data.
4. Remove supernatant and resuspend the cells at a density of 1 × 10 6 cells/ml with pre-warmed complete RPMI 1640 medium at 37°C.

Cell counting is described in Current Protocols article Strober (2001).
5. Place 3-cm 2 circular glass coverslips into wells of a 6-well plate.
6. Add 3 ml of the peritoneal cell suspension to each well and incubate for 2 hr at 37°C in an incubator.
This step allows the macrophages to adhere to the glass cover slips.
7. Gently swirl the plates and then aspirate medium.
The swirling will detach loosely adhering cells, but not firmly adhering macrophages, to get rid of cells other than macrophages. Work quickly and make sure that the coverslips are still covered by RPMI medium, to avoid cell damage.

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Current Protocols in Immunology 10. Pipet 350 μl of D-PBS into a 0.5-ml microcentrifuge tube. Add 0.7 μl of 5 mM Fluo-4AM stock solution, resulting in a 10 μM working solution.
Avoid light exposure of the Fluo-4AM solution by wrapping the microcentrifuge tube with aluminum foil.
11. Remove the glass coverslip with forceps and transfer it into an unused well of the 6-well plate.
12. Place 300 μl of the Fluo-4AM working solution on top of the coverslip.
One coverslip can be loaded with as much as 500 μl of the Fluo-4AM working solution, but covering the slide with 300 μl is sufficient.
13. Keep the coverslip in the dark at room temperature for 30 min.
14. Take the coverslip out of the 6-well plate using forceps and discard the excess fluid by blotting it on a clean paper towel.
15. Transfer the coverslip into a 50-ml centrifuge tube filled with 40 ml of D-PBS.
During the washing step, continue to hold the coverslip with the forceps.
16. Transfer the coverslip into a fresh well of the 6-well plate containing 5 ml D-PBS and incubate for 30 min at 37°C in the dark. 17. Take the coverslip out using the forceps and blot the excess fluid again with a paper towel 18. Insert the coverslip into the glass coverslip holder of an immunofluorescence or confocal microscope with a 40× objective, and add 463 μl D-PBS.
19. Set the focus using the polarized filter and analyze your sample in the FITC (Fluo-4AM) and PE channel (tdTomato).

Take a picture.
Make sure that the focus is properly set by visualizing the tdTomato signal. This picture serves as the pretreatment control.
21. Carefully add 37 μl of the hC3a working solution (see step 6 of Basic Protocol 5) to the cells.
This results in a final hC3a concentration of 37 nM.
22. Record a second picture 6 s after the addition of hC3a.
23. Process and analyze images using the data analysis software of your microscope. Figure 11B shows the microscopic evaluation of the C3a-mediated change of [Ca 2+ ] i in thioglycolate-elicited PMs from wild-type and tdTomato-C3ar1 fl/fl mice.

C5aR2-DRIVEN IFN-γ PRODUCTION FROM NK CELLS
C5aR2 expression frequently overlaps with C5aR1 expression, making it difficult to differentiate between C5aR1 and C5aR2 effects in response to C5a stimulation. One solution to this problem is to use the recently described specific C5aR2 agonist P32 (Croker et al., 2016). Another option is to use cell types that exclusively express C5aR2. Using floxed tdTomato-C5aR2 knockin mice, we uncovered that some naïve NK cells from Laumonnier et al.

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Current Protocols in Immunology spleen and blood show this property and express C5aR2 but not C5aR1 . Also, naïve B cells from blood and spleen express C5aR2 but not C5aR1. Thus, NK cells are a perfect tool to study the function of C5aR2 independently of C5aR1. NK cells are innate immune cells that exert different effector functions. In addition to their cytotoxic effects critical for killing of tumor cells, they can produce a wide array of cytokines that play important roles in viral infections and the regulation of adaptive immune responses including IFN-γ, TNF-α, and IL-22 (see Current Protocols article: Zamora, Grossenbacher, Aguilar, & Murphy, 2015). In Basic Protocol 6, we therefore describe C5aR2-driven suppression of IL-12/IL-18-induced IFN-γ production using P32 as an example of C5aR2-mediated function in NK cells.
9. Collect the supernatant and measure IFN-γ concentration using a Duo-Set IFN-γ ELISA kit according to the manufacturer's instructions. Figure 12 shows the impact of C5aR2 activation on IL-12/IL-18-induced IFN-γ production from splenic NK cells.

ISOLATION OF SPLENIC NK CELLS BY FACS
Several methods have been published to isolate NK cells from spleen, some of which use selected mAbs to deplete contaminating cells such as T, B, dendritic cells, granulocytes, and monocytes by magnetic separation (see Current Protocols article: Pak-Wittel, Piersma, Plougastel, Poursine-Laurent, & Yokoyama, 2014). The advantage of magnetic isolation is that it is fast, easy to carry out, and cost-effective, and still results in a purity of up to 95%. However, to reach higher purities (up to 99%), we describe a protocol below for positive selection by FACS.  compared to incubation at 4°C. Once inside the cell, the dye will bind to intracellular amines and its fluorescent properties will be activated.

Materials
13. Add 2 μl of the Live/Dead working solution per ml cell suspension.
14. Add 2.5 μl of each of the following antibodies per ml of cells: 18. Run the cells on a cell sorter using the gating strategy outlined in Figure 13 The number of splenic NK cells is quite low. About 2% of spleen cells are NK cells. Thus, to obtain a highly pure NK cell population, the gates need to be set very conservatively to avoid contamination with other cells. The typical yield is 5 × 10 7 total cells/spleen, i.e., 1 × 10 6 NK cells/spleen.

Fc block buffer
Dilute anti-CD16/32 antibody to a final concentration of 100 μg/ml in D-PBS/BSA (see recipe).

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Current Protocols in Immunology which allowed the genetic targeting of the AT receptors. Since then, considerable work has been done to develop new tools to track, delineate, and/or block the functions of AT receptors in mice, humans, and several other species including rats, guinea pigs, and even fish (trout) or amphibia (axolotl). Among the existing tools, the generation of AT receptor knock-out mouse strains has been one of the most important achievements for studying the role of AT receptors in steady state and under pathophysiological conditions. In the past decades, several laboratories have generated such mice, which are now broadly available and used by the scientific community. Another important advancement was the generation of AT receptor−specific antibodies that helped to identify AT receptor−expressing cells by immuno(histo)chemical methods or by flow cytometry and associated techniques (e.g., live stream imaging). However, controversial results have been obtained using antibodies to determine AT receptor expression, in particular by immunohistochemical methods (Drouin et al., 2001;Quell et al., 2017;Tschernig, Kiafard, Dibbert, Neumann, & Zwirner, 2007). Monitoring mRNA expression of AT receptors in the cells of interest in addition to antibody staining, either by northern blot or by PCR, can help to test the results obtained with antibody staining. However, mRNA expression of AT receptors does not necessarily match protein expression. Furthermore, the structure of the AT receptor genes, consisting of 2 exons with most of the coding sequence located in exon 2, strengthen the need for appropriate controls to prevent unspecific amplification of genomic DNA by PCR or recognition by radioactive probes via blot hybridization.
Recently, our laboratory has developed reporter mouse strains for C5aR1, C5aR2, and C3aR to overcome these inherent problems and ease expression studies of AT receptors. During the past few years, we generated floxed GFP-C5aR1- (Karsten et al., 2015), tdTomato-C3aR - (Quell et al., 2017), and tdTomato-C5aR2-knockin mice , which allowed ourselves and more than 30 academic laboratories in the U.S., Europe, and Australia to re-evaluate AT receptor expression in many tissues under homeostatic and multiple disease conditions. Further, many conditional AT receptor knockout strains have already been generated or are currently being developed targeting several professional and non-professional immune cells. We expect that these mice will further broaden our understanding of the multiple roles of the AT receptors in allergy, autoimmunity, infection, cancer, metabolic disease, transplantation, ischemia reperfusion injury, hypertonia and several other diseases or disorders.

Working with anaphylatoxins
One critical issue in handling ATs is their tendency to bind to plastic surfaces such as polypropylene walls of microcentrifuge tubes due to their basic nature. To avoid absorption to plastic surfaces, use low-retention tubes as well as low-retention pipet tips whenever you use pure AT solutions. To achieve this, several companies provide special tubes with a highly polished surface. Further, it is important to avoid repeated freezing/thawing processes.

Analysis of C5aR1-driven ERK1/2 phosphorylation in GFP-C5aR1 + cells
For staining of intracellular proteins using methanol-mediated fixation and permeabilization, the centrifugation steps and the handling of cells after treatment are critical. Methanol treatment makes the cells very fragile and sensitive to centrifugal forces. Thus, handle cells with care during washing and in particular the centrifugation steps. Because methanol-treated cells change buoyancy, the pellet is very fragile, unstable and difficult to spin down. Do not exceed 500 × g in your centrifugation steps.

C3a-induced increase in intracellular Ca 2+
Depending on the cell type, you have to consider use of D-PBS supplemented with Ca 2+ when measuring C3a-induced increase in [Ca 2+ ] i . In contrast to C5aR1 activation, which mobilizes intracellular Ca 2+ stores of the endoplasmic reticulum (Norgauer et al., 1993), C3aR pathway activation requires uptake of extracellular calcium in human neutrophils (Norgauer et al., 1993). In some cells, C3aR activation drives the mobilization of intracellular calcium from intracellular stores as in transfected cell lines (Chao et al., 1999) or astrocytes (Sayah et al., 2003). When the C3aR signaling pathway has not been evaluated yet in the cell type under investigation, we recommend using D-PBS supplemented with Ca 2+ .

Determination of AT receptor expression using antibodies
Several polyclonal and monoclonal antibodies have been reported that have been used Laumonnier et al.

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Current Protocols in Immunology to determine C5aR1, C5aR2, or C3aR expression in immune and tissue cells (Laumonnier et al., 2017). Unfortunately, many of the available AT receptor antibodies not only recognize the AT receptors, but can also bind additional structures and/or epitopes . Thus, we recommend using AT receptor−deficient mice or additional PCR measurements to control for the specificity of a given AT receptor antibody staining, and not rely solely on fluorescence minus one (FMO) controls. In our hands, C5aR1 mAb 20/70 shows high specificity for C5aR1, and frequently matches results obtained with GFP-C5aR1 reporter mice. For C5aR2, mAb 468705 is widely used, but we found that this mAb not only stained bone marrow-derived neutrophils and macrophages as well as peritoneal macrophages from the tdTomato-C5ar2 fl/fl mice, but also those from C5ar2 -/-, suggesting that it cross reacts with other structures on these cells . However, this antibody was found to specifically stain C5aR2 in endothelial cells (Miyabe, Miyabe, Mani, Mempel, & Luster, 2019), indicating that the specificity of this antibody is dependent on the cell type used. Regarding C3aR, we found that the anti-C3aR clone 14D4 matched the results obtained with floxed tdTomato-C3aR knock-in mice. In contrast, we found positive staining in different immune cells from C3ar1 -/mice using antibodies widely used in the past to stain C3aR, i.e., the D12, D20, and H300 clones (Quell et al., 2017) (Fig. 14).

Impact of fixatives on fluorochrome stability
Several fluorochromes (such as PE, PerCP), their respective tandem dyes (such as PE-Cy7 or PercP-Cy5.5), and to a certain extent APC, are sensitive to methanol treatment and lose their structural integrity and hence their capacity to emit light. Because of their instability, we do not recommend use of such dyes prior to methanol treatment but advise the reader to consider alternative fluorochromes for Basic Protocol 4. APC might be used when the expression of the target molecule/receptor is high.

Genotyping of floxed AT receptor reporter mice
For good lab practice, include water controls, internal controls, i.e., primers targeting the wild-type gene in the PCR reaction and run reference lanes for wild-type, heterozygous, and homozygous genotypes with each experiment, as shown in the examples in Figures 2, 3, 6 and 7.

Strength of the reporter molecules
Due to the fact that the GFP protein emits quite strongly, the signal can be easily observed in cells expressing high levels of C5aR1 such as neutrophils using Figure 14 Binding of different anti-C3aR antibodies to BALB/c wild-type, tdTomato-C3ar1 fl/fl , and C3ar1 -/mice. The histograms show C3aR surface expression in F4/80 + peritoneal macrophages from wild-type, tdTomato-C3ar1 fl/fl , and C3ar1 -/mice using C3aR-antibody clones 14D4 and D20. The gray histogram depicts the C3aR signal obtained from wild-type mice; the black line shows the signal from tdTomato-C3ar1 fl/fl , and the dashed line shows the signal from C3ar1 -/mice.

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Current Protocols in Immunology either homozygous or heterozygous C5aR1 reporter mice. The use of heterozygous mice should be preferred whenever the full functionality of the AT receptor is required, since we have shown previously that, although still functional, homozygous cells react somewhat more weakly to C5a than wild-type or heterozygous cells . Interestingly, heterozygous animals can also be used to investigate the expression of C5aR1 in cells with high autofluorescence, such as alveolar macrophages (Fig. 15). Although very useful, a couple of things have to be considered regarding GFP as a reporter fluorochrome. It is well appreciated that it exerts high immunogenicity and cytotoxicity, and there is phototoxicity associated with the blue light used to excite GFP (Ansari et al., 2016). Also, GFP is sensitive to fixation by formaldehyde or ethanol dehydration (Jockusch, Voigt, & Eberhard, 2003). In contrast, the tdTomato protein is more stable in this regard (Shaner, Steinbach, & Tsien, 2005). It is well suited for immunofluorescence microscopy (Fig. 11B, [Morris, Klanke, Lang, Lim, & Crombleholme, 2010], [Quell et al., 2017]) without extensive optimization (Zhanmu et al., 2019). Furthermore, tdTomato emits more strongly than most other chromophores (Shaner et al., 2004;Shaner et al., 2005) making it a very in-teresting surrogate for cells with minor expression of the target molecule.

Flow cytometer settings used with AT receptor reporter mice
GFP is excited by the blue laser (488 nm) with an emission maximum at 509 nm. Therefore, GFP is typically detected in the FITC channel (530/25). Using a narrower-bandpass filter (510/12) that is closer to the emission maximum can improve the detection of dimly expressed GFP. Further, tdTomato is best excited by the yellow-green laser (561 nm), and is typically detected in the PE channel using a 585/15 bandpass filter. Consequently, antibodies labeled with fluorochromes that are detected in the FITC channel cannot be used with the GFP-C5aR1 reporter mouse, nor can antibodies labeled with fluorochromes detected in the PE channel with tdTomato-C3aR and tdTomato-C5aR2. Depending on the signal intensity of the fluorescent proteins, it is recommended to minimize the spillover into their detection channels. Autofluorescence from cells like macrophages, due to their high metabolic activity involving NADPH, might be a source of false positive signals in GFP and tdTomato detection channels. Therefore, when working with highly autofluorescent cells (like macrophages), Figure 15 Comparison of the GFP signal in wild-type, GFP-C5ar1 fl/+ and GFP-C5ar1 fl/fl mice in alveolar macrophages with strong autofluorescence. Histogram showing the GFP expression in SiglecF + CD11c + alveolar macrophages from wild-type (gray histogram), GFP-C5ar1 fl/+ mice (light green), and GFP-C5ar1 fl/fl mice (dark green). Data are representative of three independent experiments. Laumonnier et al.

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Current Protocols in Immunology fluorochromes emitting in the FITC or PE channel, but also emitting in the violet channels (BV421, 450, and 510), should be used with care.