Flow cytometry Anti-bodies Human - CD22

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 3 matching solutions for this experiment

Purified Mouse Anti-Human CD22

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Company recommended amount of respective antibodies, and 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min.	The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired.

Protocol tips
Company recommended amount of respective antibodies, and 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min.
Downstream tips
The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired.

Manufacturer protocol Publication protocol 1 Relevant paper

FITC Mouse Anti-Human CD22

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Live cells were identified using LIVE/DEAD Fixable Near-IR staining (Molecular Probes) according to the manufacturer’s instructions. Cultured cells were pelleted, washed, and stained with LIVE/DEAD, followed by surface staining with fluorescently labeled mAbs	Multicolor flow cytometry was performed using a five-laser LSRII flow cytometer (BD) and analyzed with FlowJo software (Tree Star).

Protocol tips
Live cells were identified using LIVE/DEAD Fixable Near-IR staining (Molecular Probes) according to the manufacturer’s instructions. Cultured cells were pelleted, washed, and stained with LIVE/DEAD, followed by surface staining with fluorescently labeled mAbs
Downstream tips
Multicolor flow cytometry was performed using a five-laser LSRII flow cytometer (BD) and analyzed with FlowJo software (Tree Star).

Manufacturer protocol Publication protocol 1 Relevant paper

CD22-PE, SJ10.1H11, 2 mL, ASR

Beckman Coulter

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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