The fluoresence activated cell sorting (FACS) screen examines the major hematopoietic cell subsets in the blood in response to MCMV infection.

Materials:

• Syringes
• MiniCollect^® Blood Collection Tube (EDTA coated)
• Falcon^® 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap

Reagents:

• MCMV stock
• Red blood cell (RBC) lysis buffer
• FACS buffer: 1% FCS/BSA in PBS
• Antibodies

Infection

1. Inject MCMV (1.5 x 10⁵ PFU per 20 g) i.p. into the screening mice. Return animals to housing.

Blood Sample Preparation for FACS analysis

2. Five days post-MCMV infection, collect approximately 100 μL of blood from each mouse in MiniCollect^® blood collection tubes.

3. Add 1 mL of RBC lysis buffer into the tubes and transfer samples to Falcon^® 5 mL round-bottom test tubes with cell strainer cap.

4. Add another 1 mL of RBC lysis buffer, mix, and incubate for 5-10 minutes.

5. Add 2 mL of FACS buffer into the test tubes, centrifuge at 500 x g for 5 minutes, and discard the supernatants.

6. Resuspend cell pellets with 50 μL of 1:200 Fc block in FACS buffer, and incubate for 15 minutes at 4°C.

7. Add 50 μL of antibody cocktail including anti-CD3ε, CD4, CD8a, B220, CD19, NK-1.1, F4/80, CD11b, CD11c, CD62L, CD44, CD43, CD5, IgD, IgM (1:100-1:300 in FACS buffer; Table 1), and incubate for 30 minutes at 4°C.

8. Wash the cells by adding 4 mL of FACS buffer and centrifuging at 500 x g for 5 minutes, discard the supernatants.

9. Resuspend the cells in 250 μL FACS buffer and analyze on a flow cytometer.

Table 1. Antibodies used in post-MCMV FACS screen