CD122 antibody | TU27

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For intracellular FACS-staining of PMNs in whole blood, the intracellular proteins were blocked by adding 10 μg of Brefeldin A / ml to whole blood for about 4 hours at 37°C/5% CO2. The permeability of the cell membrane was increased by adding 500 μl 1 × FACS permeabilizing solution. Cells were washed with FACS buffer + Saponin 0.2% and stained with 2 mg of anti-CD122-FITC.	Cells were washed three times with 2 ml FACS buffer + Saponin 0.2%, fixed by 300 μl of 1% PFA and analyzed by FACSCalibur and CellQuest software (Becton-Dickinson, Heidelberg, Germany).

Protocol tips
For intracellular FACS-staining of PMNs in whole blood, the intracellular proteins were blocked by adding 10 μg of Brefeldin A / ml to whole blood for about 4 hours at 37°C/5% CO2. The permeability of the cell membrane was increased by adding 500 μl 1 × FACS permeabilizing solution. Cells were washed with FACS buffer + Saponin 0.2% and stained with 2 mg of anti-CD122-FITC.
Downstream tips
Cells were washed three times with 2 ml FACS buffer + Saponin 0.2%, fixed by 300 μl of 1% PFA and analyzed by FACSCalibur and CellQuest software (Becton-Dickinson, Heidelberg, Germany).

Publication protocol

For intracellular FACS-staining of PMNs in whole blood, the intracellular proteins were blocked by adding 10 μg of Brefeldin A / ml to whole blood for about 4 hours at 37°C/5% CO2. The permeability of the cell membrane was increased by adding 500 μl 1 × FACS permeabilizing solution. Cells were washed with FACS buffer + Saponin 0.2% and stained with 2 mg of anti-CD122-FITC. Anti-mouse IgG-FITC and anti-CD66-FITC were used as a positive and negative control, respectively. FITC conjugated antibodies mixed well and incubated as mentioned above. Cells were washed three times with 2 ml FACS buffer + Saponin 0.2%, fixed by 300 μl of 1% PFA and analyzed by FACSCalibur and CellQuest software (Becton-Dickinson, Heidelberg, Germany). Results are expressed as a percentage of positive cells Mean Fluorescence Intensity (MFI) in the respective gate.

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