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Purpose
This test differentiates immune cell sub-populations via flow cytometry in one panel.
Experimental Design
- Minimum number: 7M + 7F
- Age at test: Week 16
- Sex: Both (sexually dimorphic)
Equipment
Reagents and equipment for sample preparation
- Spleen Dissociation Kit (Miltenyi Biotec)
- gentleMACS Dissociator (Miltenyi Biotec)
- gentleMACS C Tubes (Miltenyi Biotec)
- FACS buffer: 2% Fetal Bovine Serum, 10mM HEPES, 2mM EDTA in HBSS without Ca2+ and Mg2+
- ACK Lysis buffer: 150mM NH4Cl, 10mM KHCO3, 0.1mM Na2EDTA in H2O
- CellTrics filters 100μm (Sysmex)
- BD Horizon Brilliant Stain Buffer Plus
- Anti-CD16/CD32 antibody - Fc block (BD Biosciences)
Procedure
Sample preparation
- Dissociate spleens enzymatically in gentleMACS C Tubes according to the Spleen Dissociation Kit protocol.
- Fill the tubes with cell suspensions up to 10 mL with FACS buffer and transfer the suspensions through 100 μm cell strainer into 15mL tubes.
- Centrifuge samples at 400 g for 8 minutes at 4°C.
- Resuspend pellets in 1 mL of ACK Lysis buffer and incubate for 3 minutes at room temperature.
- Add FACS buffer up to 5 mL and filter cell suspension through the 100 μm cell strainer.
- Count total number of live cells using Trypan Blue stain and Cellometer Auto T4 with Nexcelom cell counting chambers.
- Transfer 2.5x106 of live cells per well into a 96-well U bottom plate. Centrifuge plate at 400 g for 4 minutes at 4°C.
- Discard supernatant and proceed with staining.
- Resuspend pellets in 50 μL of staining cocktail (antibodies including Fc block (1:200) in FACS buffer containing 20% of Brilliant stain buffer):
-
Specificity Fluorochrome Clone Catalog # Manufacturer Dilution CD8a BUV395 53-6.7 563786 BD Biosciences 1:200 CD4 BUV496 RM4-5 741050 BD Biosciences 1:400 F4/80 BUV563 T45-2342 749284 BD Biosciences 1:400 CD19 BUV615 1D3 751213 BD Biosciences 1:800 γδTCR BUV661 GL3 750410 BD Biosciences 1:200 CD21/CD35 BUV737 7G6 612810 BD Biosciences 1:2000 CD44 BUV805 IM7 741921 BD Biosciences 1:400 CD5 BV421 53-7.3 562739 BD Biosciences 1:400 Viability SYTOX Blue - S34857 Invitrogen 1:20000 CD11c BV605 N418 117334 BioLegend 1:400 Ly6G BV650 1A8 740554 BD Biosciences 1:400 BST2 (CD317) BV711 927 747604 BD Biosciences 1:400 Ly6C FITC AL-21 553104 BD Biosciences 1:800 MHCII BB630 M5/114.15.2 624294 BD Biosciences 1:800 KLRG1 BB700 2F1 742199 BD Biosciences 1:400 CD49d BB790 R1-2 624296 BD Biosciences 1:400 CD23 PE B3B4 101607 BioLegend 1:800 CD161 PE-CF594 PK136 562864 BD Biosciences 1:800 CD25 PE-Cy7 PC61 552880 BD Biosciences 1:400 GITR (CD357) APC DTA-1 624346 BD Biosciences 1:800 CD11b APC-R700 M1/70 564985 BD Biosciences 1:2000 CD62L APC-Cy7 MEL-14 560514 BD Biosciences 1:2000
- Incubate samples for 30 minutes in dark on ice.
- Wash samples with 200 μL of FACS buffer and centrifuge for 4 minutes at 400 g at 4 °C.
- Discard supernatant and resuspend pellets in 150 μL of FACS buffer containing SYTOX Blue (diluted 1:40 000).
- Acquire 500 000 live cells.
Notes
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