Protocols


Screen (pdf version)
ScreenFACS screen after MCMV infection
Posted On03/17/2017 3:20 PM
AuthorTao Yue, Duanwu Zhang
Background

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

Reagents and Solutions

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
Method

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

Antibody Format Dilution Readout
anti-CD3ε FITC 1:100 CD3+ T cells
anti-CD4 BV786 1:200 CD4T cells
anti-CD8a BV510 1:200 CD8+ T cells and DCs
anti-B220 AF700 1:150 B cells
anti-CD19 BUV395 1:300 B cells
anti-NK-1.1 BV650 1:200 NK-1.1+ natural killer cells
anti-F4/80 PE 1:150 Macrophages
anti-CD11b BV605 1:300 Macrophages/neutrophils
anti-CD11c BV711 1:200 CD11c​DCs
anti-CD44 PE-CF594 1:200 lymphocyte activation, effector-memory T cells
anti-CD62L PE-Cy7 1:300 Naive/central memory vs effector memory
anti-CD43 APC 1:300 B1 vs B2 cells
anti-CD5 BV421 1:300 B1a vs B1b cells
anti-IgD APC-Cy7 1:300 IgD+ B cells
anti-IgM PerCP-Cy5.5 1:300 IgM+ B cells
Edit History
Duanwu Zhang 2019-06-05 5:56 PM (current)
Duanwu Zhang 2019-06-05 5:18 PM
Anne Murray 2017-03-17 3:20 PM