Flow cytometry Anti-bodies Human - CD122

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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Found 2 matching solutions for this experiment

CD122 antibody | TU27

Bio-Rad Laboratories

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).

Manufacturer protocol Publication protocol 1 Relevant paper

PE Mouse Anti-Human CD122

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA)

Protocol tips
Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA)

Manufacturer protocol Publication protocol 1 Relevant paper

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