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:
-
Collect
-
200,000 Jurkat cells (previously maintained in RPMI1660 10%FBS) per FACS tube
-
Anti-IFNAR2 MAb (PBL Catalog No. 21385-1)
-
Isotype-matched negative control MAb (mouse IgG2a, e.g., DakoCytomation (X0943).
-
Wash cells twice in wash buffer. (Resuspend gently, then centrifuge at 4°C, 1800 rpm 5 min).
-
Carefully pipette off the supernatant and discard.
-
Gently mix to resuspend cells in 50 μl wash buffer.
-
Add the MAbs.
-
Control MAb tube: 3 μl negative control mouse IgG2a (DakoCytomation)
-
IFNAR2 MAb tube: Anti-IFNAR2 MAb (PBL 21385-1) to a final concentration of 2.5 μg/ml
-
Incubate on ice for 40 min.
-
Wash twice in wash buffer as above.
-
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.
-
Incubate on ice for 40 min.
-
Wash twice in wash buffer.
-
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.
-
Incubate on ice for 10 min.
-
Wash cells twice in wash buffer.
-
Resuspend cells in PBS plus 1 μg/ml propidium-iodine (to exclude dead cells from the measurements. This can be omitted).
-
Harvest cells for FACS.
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:
-
Collect
-
200,000 Jurkat cells (previously maintained in RPMI1660 10%FBS) per FACS tube
-
Anti-IFNAR2 MAb (PBL Catalog No. 21385-1)
-
Isotype-matched negative control MAb (mouse IgG2a, e.g., DakoCytomation (X0943).
-
-
Wash cells twice in wash buffer. (Resuspend gently, then centrifuge at 4°C, 1800 rpm 5 min).
-
Carefully pipette off the supernatant and discard.
-
Gently mix to resuspend cells in 50 μl wash buffer.
-
Add the MAbs.
-
Control MAb tube: 3 μl negative control mouse IgG2a (DakoCytomation)
-
IFNAR2 MAb tube: Anti-IFNAR2 MAb (PBL 21385-1) to a final concentration of 2.5 μg/ml
-
-
Incubate on ice for 40 min.
-
Wash twice in wash buffer as above.
-
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.
-
Incubate on ice for 40 min.
-
Wash twice in wash buffer.
-
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.
-
Incubate on ice for 10 min.
-
Wash cells twice in wash buffer.
-
Resuspend cells in PBS plus 1 μg/ml propidium-iodine (to exclude dead cells from the measurements. This can be omitted).
-
Harvest cells for FACS.
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