Blood cell suspensions were prepared as described above and incubated with the FC blocking antibody α-CD16/32 (eBioscience, San Diego, CA; [5 μg/ml]) for 10 min. The cells were subsequently incubated with the following antibody panel for 25 mins. on ice: α-F4/80-AF488 (AbD serotec, Oxford, UK; [0.5 μg/ml]), α-CD115-PE (Biolegend, San Diego, CA; [1 μg/ml]), α-CD11b-PE/Cy7 (Biolegend; [0.5 μg/ml]), α-Ly6C-PerCP/Cy5.5 (BioLegend; [0.5 μg/ml]), α-Ly6G-APC (BioLegend; [1 μg/ml]), α-CD3e-APC (eBioscience; [1 μg/ml]), α-CD19-APC (eBioscience; [1 μg/ml]), α-CD49b-APC (eBioscience; [1 μg/ml]), and DAPI (Sigma; [0.1ug/ml]). Samples were stained concurrently with fluorescence minus one (FMO) antibody panels. Following a series of washes, the cells were resuspended in MACS buffer, and sorted on the BD Influx Cell Sorter (Becton, Dickinson and Company, Franklin Lakes, NJ). FlowJo software (Tree Star Inc., v.9.2, Ashland, OR) was used for data analysis.
αcd16 32
Manufactured by Thermo Fisher Scientific
αCD16/32 is a laboratory product manufactured by Thermo Fisher Scientific. It is a primary antibody used for the detection and analysis of the CD16 and CD32 cell surface antigens in flow cytometry and other immunoassay applications.
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Lab products found in correlation
Influx cell sorter, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
α cd11b pe cy7, by BioLegend (1 mentions)
α ly6g apc, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Efluor450 viability dye, by Thermo Fisher Scientific (1 mentions)
Onecomp ebeads, by Thermo Fisher Scientific (1 mentions)
Anti mouse ly6g fitc, by Thermo Fisher Scientific (1 mentions)
100 m cell strainer, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Mhc class 2 1 a 1 e, by Thermo Fisher Scientific (1 mentions)
Facsdiva 6, by BD (1 mentions)
Gentlemacs c tube, by Miltenyi Biotec (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Moflo cell sorter, by Beckman Coulter (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
5 protocols using αcd16 32
1
Multicolor Flow Cytometry for Immune Cell Profiling
2
Isolation and Phenotyping of Mouse Peritoneal Cells
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Mouse peritoneal cells were collected via a peritoneal cavity wash with 10 mL of PBS-5% FCS. Single-cell suspensions were prepared in FACS buffer (PBS + 0.5% BSA + 2 mM EDTA). Fc receptors were blocked with αCD16/32 (1/40 dilution, ref: 14-0161-82, eBioscience) prior to the application of the following cocktail: viability dye eFluor450 (ref: 65-0863-14, eBioscience), anti-mouse SiglecF-APC (dilution 1/40, clone S17007L, ref: 155508, Biolegend) and anti-mouse Ly6G-FITC (dilution 1/40, clone RB6-8C5, ref: 11-5931-82, eBioscience) or their matched isotype controls using a fluorescence-minus-one method. Samples were fixed in FACS buffer containing 0.5% PFA and shipped at 4 °C to UK. All multi-labelled cell samples were subsequently acquired using a BD LSR II flow cytometer (BD Bioscience) and analysed on FlowJo Software (Supplementary Fig. 7 ). OneComp eBeads (eBioscience) were used to optimise antibody staining panels and apply compensation. For compensation controls, optimal PMT voltages for the positive signal to be detected were set within 104 and 105 whereas negative signal was set to below 102.
3
Comprehensive Immune Cell Profiling
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Splenocytes or lymph node cells were extracted into single cell suspension in 1× PBS, and RBCs lysed by incubation in ACK lysis buffer (Biofluids), single cell suspensions were passed through 100 µm cell strainer (BD Falcon). Cells were washed and FcγRII/III blocked with αCD16/32 (eBiosciences). Surface markers were stained with fluorochrome‐conjugated mAbs toCD3, CD4, CD69, CCR7, IL7Rα, CD62L, CD44, CD45, CD103, CD25, CD8α, CD11b, CD11c, CD19, CD62L, CD80, CD86, CD117 (c‐kit), DX5, F4/80, MHC Class II (I‐A/I‐E), (eBiosciences, BD Pharmingen, Biolegend, AbD Serotec). Treg cells were further stained by intracellular staining of Foxp3 with a kit according to manufacturer's instructions (eBioisciences). Samples were acquired on the LSR II quad‐laser cytometer running FACSDiva (BD Immunocytometry).
4
Flow Cytometry Analysis of Mouse Immune Cells
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For flow cytometry analysis, single cell suspensions were made from mouse spleen by gentle dissociation or from mouse hearts by perfusing for 3 min with 1× PBS + 0.5% FBS, and digested in gentleMACS C Tubes according to manufacturer’s instructions (Miltenyi Biotec). Viability was determined by LIVE/DEAD staining according to manufacturer’s instructions (Life Technologies). Cells were blocked with α-CD16/32 (eBioscience), and surface markers were stained with fluorochrome-conjugated mAbs (eBioscience, BD, and BioLegend). Samples were acquired on the LSR II cytometer running FACSDiva 6 (BD). Data were analyzed with FlowJo 7.6 (Tree Star). For FACS isolation, single cell suspension from mouse heart or spleen was first purified with a 20–80% Percoll (GE Healthcare) gradient to eliminate dead cells and debris. Cells were then stained with fluorochrome-conjugated mAbs (eBioscience, BD, and BioLegend) and sorted with a MoFlo Cell Sorter (Beckman Coulter).
5
Multiparametric Flow Cytometry Analysis
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Single cell suspensions were prepared in FACS buffer (PBS+0.5%BSA+2mMEDTA). Fc receptors were blocked with αCD16/32 (eBioscience). Live/dead cell differentiation was undertaken with fixable viability dye efluor 450 as per manufacturer’s instructions (eBioscience). Cell staining was undertaken utilising specific labelled anti-mouse antibodies or their matched isotype controls using a fluorescence-minus-one method. Intracellular staining was done following permeabilisation buffer treatment (eBioscience). using a zenon Alexa Fluor 488 Rabbit IgG labelling kit as per manufacturer’s instructions (Invitrogen). All multi-labelled cell samples were subsequently acquired using a BD LSR II flow cytometer (BD Bioscience) and analysed on FloJo Software (S9 –S11 Figs; also see supplementary methods). OneComp eBeads were used to optimise antibody staining panels and apply compensation. For compensation controls, we applied optimal PMT voltages for the positive signal to be detected within 10^4 and 10^5 whereas negative signal set to be below 10^2. Compensation matrices were applied in which there was <40% overlap in any signal combination.
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