Flow cytometry Anti-bodies Human - CD20

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

CD20 V450

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	. After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences.	A total of 200,000 events were acquired for assessment of these investigated antibodies. Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non‐viable cells, debris, and aggregates were excluded based on forward scatter‐height/forward scatter‐area (FSC‐H/FSC‐A).

Protocol tips
. After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences.
Downstream tips
A total of 200,000 events were acquired for assessment of these investigated antibodies. Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non‐viable cells, debris, and aggregates were excluded based on forward scatter‐height/forward scatter‐area (FSC‐H/FSC‐A).

Manufacturer protocol Publication protocol 1 Relevant paper

FITC anti-human CD20 Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Cells were stained with a fixable Live/Dead dye (ThermoFisher Scientific) concurrently with saturating concentrations of surface mAbs (Table S2) in a buffer containing 50%ABrilliant Violet Buffer (BD Bioscience) and 50%APBS for 30min at 4°C. Once stained PBMC and IHL were fixed and permeabilised, where necessary, with either Cytofix/Cytoperm (BD Bioscience), or with the FoxP3 Buffer Kit (BD Bioscience) according to manufacturer’s instructions.	All samples were acquired in a 0.1% saponin buffer on an X20FortessaASORP (BD Biosciences) and analysed in FlowJo (TreeStar).

Protocol tips
Cells were stained with a fixable Live/Dead dye (ThermoFisher Scientific) concurrently with saturating concentrations of surface mAbs (Table S2) in a buffer containing 50%ABrilliant Violet Buffer (BD Bioscience) and 50%APBS for 30min at 4°C. Once stained PBMC and IHL were fixed and permeabilised, where necessary, with either Cytofix/Cytoperm (BD Bioscience), or with the FoxP3 Buffer Kit (BD Bioscience) according to manufacturer’s instructions.
Downstream tips
All samples were acquired in a 0.1% saponin buffer on an X20FortessaASORP (BD Biosciences) and analysed in FlowJo (TreeStar).

Manufacturer protocol Publication protocol 1 Relevant paper

PE Mouse Anti-Human CD20

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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