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. 

Flow cytometric analysis of Jurkat cells

Figure 1. Flow cytometric analysis of Jurkat cells with expression of IFNAR2 shown in the green open histogram and the background of mouse IgG2a negative control in the violet histogram. Boxed results show percentage of positive cells and mean specific fluorescence, calculated by subtracting the positivity and the mean of fluorescence obtained with isotype-matched control Ig from that detected with the specific mAb.

 

Author:

Courtesy of Dr. Laura Conti, Center for Experimental Research and Medical Studies (CERMS), San Giovanni Battista Hospital, Turin, Itlay

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