Flow Cytometry For IFNAR2 On JURKAT Cells

Protocol for Flow Cytometric Analysis of IFNAR2 Surface Expression on JURKAT Cells

 

Materials:

Phosphate buffered saline (PBS)

Wash buffer: PBS containing 0.5% BSA and 0.02% NaN₃

 

Procedure:

1. Collect

   1. 200,000 Jurkat cells (previously maintained in RPMI1660 10%FBS) per FACS tube

   2. Anti-IFNAR2 MAb (PBL Catalog No. 21385-1)

   3. Isotype-matched negative control MAb (mouse IgG2a, e.g., DakoCytomation (X0943). 

2. Wash cells twice in wash buffer. (Resuspend gently, then centrifuge at 4°C, 1800 rpm 5 min). 

3. Carefully pipette off the supernatant and discard. 

4. Gently mix to resuspend cells in 50 μl wash buffer. 

5. Add the MAbs. 

   1. Control MAb tube: 3 μl negative control mouse IgG2a (DakoCytomation)

   2. IFNAR2 MAb tube: Anti-IFNAR2 MAb (PBL 21385-1) to a final concentration of 2.5 μg/ml

6. Incubate on ice for 40 min. 

7. Wash twice in wash buffer as above. 

8. Resuspend cells in 50 μl wash buffer with biotin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch (115-066-072) to a final concentration of 5 μg/ml. 

9. Incubate on ice for 40 min. 

10. Wash twice in wash buffer. 

11. Resuspend cells in 50 μl wash buffer with R-Phycoerythrin-conjugated Streptavidin (Jackson ImmunoResearch (016-110-084) to a final concentration of 2.5 μg/ml. 

12. Incubate on ice for 10 min. 

13. Wash cells twice in wash buffer. 

14. Resuspend cells in PBS plus 1 μg/ml propidium-iodine (to exclude dead cells from the measurements. This can be omitted). 

15. Harvest cells for FACS.