Materials:
-
Appliable stimulated and un-stimulated cells
-
1X PBS pH 7.2
-
Bovine Serum Albumin (BSA)
-
Normal human serum
-
5 ml polystyrene tubes; BD Falcon Catalog No. 352052
-
Isotype control: PE-Mouse IgG1 kappa; BD Pharmingen Catalog No. 554680
Method:
-
Collect the cells in 15 ml conical tubes and spin for 5 minutes at 1000-1200 rpm.
-
Wash the cells 1X with PBS, spin at 1000-1200 rpm for 5 minutes.
-
Re-suspend the cells in 10% normal human serum to block Fc receptors. Allow to sit on ice for 30 minutes.
-
Wash the cells 1X with PBS, spin at 1000-1200 rpm
-
Re-suspend each cell pellet in a sufficient volume of PBS/0.1% BSA to allow for 100 μl cell volume per sample and per isotype control. Also be careful the re-suspended cells are neither too dilute nor too concentrated.
-
Prepare 5 μg/ml, 2.5 μg/ml and 1.25 μg/ml of the labeled antibody to be tested in PBS/0.1% BSA (if an optimal antibody concentration has been previously established in your system, a titration is not required).
-
Add 10 μl of each antibody concentration to empty BD Falcon polystyrene tubes in duplicates 9one dedicated for stimulated cells and the other for un-stimulated cells).
-
Add 10μl of isotype control to two tubes labeled as positive isotype control and negative isotype control. Note: For optimal results dilute the isotype control to the same concentration as the labeled antibody you are testing. In case of multiple dilutions of the antibody it is suggested to dilute the isotype to the mean of the concentrations.
-
Add 100 μl of cells from step 5 to the tubes containing the antibody and the isotype controls. Mix well and incubate for 30 minutes in the dark at room temperature.
-
Wash cells 2X with PBS/0.1% BSA, spin at 1000-1200 rpm for 5 minutes.
-
Re-suspend the cells in 500 μl PBS.
-
Store cells at 2-8°C in the dark prior to evaluation by Flow Cytometry. The cells should be evaluated by Flow Cytometry within 24 hours
Materials:
-
Appliable stimulated and un-stimulated cells
-
1X PBS pH 7.2
-
Bovine Serum Albumin (BSA)
-
Normal human serum
-
5 ml polystyrene tubes; BD Falcon Catalog No. 352052
-
Isotype control: PE-Mouse IgG1 kappa; BD Pharmingen Catalog No. 554680
Method:
-
Collect the cells in 15 ml conical tubes and spin for 5 minutes at 1000-1200 rpm.
-
Wash the cells 1X with PBS, spin at 1000-1200 rpm for 5 minutes.
-
Re-suspend the cells in 10% normal human serum to block Fc receptors. Allow to sit on ice for 30 minutes.
-
Wash the cells 1X with PBS, spin at 1000-1200 rpm
-
Re-suspend each cell pellet in a sufficient volume of PBS/0.1% BSA to allow for 100 μl cell volume per sample and per isotype control. Also be careful the re-suspended cells are neither too dilute nor too concentrated.
-
Prepare 5 μg/ml, 2.5 μg/ml and 1.25 μg/ml of the labeled antibody to be tested in PBS/0.1% BSA (if an optimal antibody concentration has been previously established in your system, a titration is not required).
-
Add 10 μl of each antibody concentration to empty BD Falcon polystyrene tubes in duplicates 9one dedicated for stimulated cells and the other for un-stimulated cells).
-
Add 10μl of isotype control to two tubes labeled as positive isotype control and negative isotype control. Note: For optimal results dilute the isotype control to the same concentration as the labeled antibody you are testing. In case of multiple dilutions of the antibody it is suggested to dilute the isotype to the mean of the concentrations.
-
Add 100 μl of cells from step 5 to the tubes containing the antibody and the isotype controls. Mix well and incubate for 30 minutes in the dark at room temperature.
-
Wash cells 2X with PBS/0.1% BSA, spin at 1000-1200 rpm for 5 minutes.
-
Re-suspend the cells in 500 μl PBS.
-
Store cells at 2-8°C in the dark prior to evaluation by Flow Cytometry. The cells should be evaluated by Flow Cytometry within 24 hours
