Flow cytometry Anti-bodies Human - CD52

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

CD52 Monoclonal Antibody (CF1D12), PE

eBioscience

Upstream tips	Protocol tips	Downstream tips
The BM aspirates were separated into 100 μL aliquots.	Fixation and permeabilization (FIX&PERM) Solutions A and B (Biozol, Eching, Germany) were used for cytoplasmic anti‐κ and anti‐λ staining, and BD FACS™ Lysing Solution was used for red cell lysis (BD, Heidelberg, Germany). Before measurement, the cells were washed twice and resuspended in 500 μL of phosphate‐buffered saline (Life Technologies, Carlsbad, California).	The measurement was performed on a FACSCanto™ II cell analyzer (BD, Heidelberg, Germany). The compensation matrix was calculated using BD CompBead particles (BD, Heidelberg, Germany), and the compensation setup tool in BD FACSDiva™ software was used. Data were analyzed using the Infinicyt™ software (Cytognos, Salamanca, Spain).

Upstream tips
The BM aspirates were separated into 100 μL aliquots.
Protocol tips
Fixation and permeabilization (FIX&PERM) Solutions A and B (Biozol, Eching, Germany) were used for cytoplasmic anti‐κ and anti‐λ staining, and BD FACS™ Lysing Solution was used for red cell lysis (BD, Heidelberg, Germany). Before measurement, the cells were washed twice and resuspended in 500 μL of phosphate‐buffered saline (Life Technologies, Carlsbad, California).
Downstream tips
The measurement was performed on a FACSCanto™ II cell analyzer (BD, Heidelberg, Germany). The compensation matrix was calculated using BD CompBead particles (BD, Heidelberg, Germany), and the compensation setup tool in BD FACSDiva™ software was used. Data were analyzed using the Infinicyt™ software (Cytognos, Salamanca, Spain).

Publication protocol 1 Relevant paper

PE Mouse anti-Human CD52

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	About 1,000,000 cells from whole blood or bone marrow samples anticoagulated with EDTA were incubated for 20 min in the dark at room temperature with each MoAb. Red blood cells were lysed by incubation with Excellyse I (EXBIO, Praha, CZ), followed by incubation with distilled water. Samples were washed and resuspended in phosphate buffered saline (PBS). All reagents were used according to the manufacturer's instructions. All samples were processed within 48 h of collection.	Immediately after preparation, samples were analyzed on a FACSCalibur flow cytometer using CellQuest™ Pro software (BD Biosciences, San Diego, CA, USA). About 100,000 events per sample were obtained. Infinicity™ Flow Cytometry version 1.7 software (Cytognos, SL, ES) was used for data analysis.

Protocol tips
About 1,000,000 cells from whole blood or bone marrow samples anticoagulated with EDTA were incubated for 20 min in the dark at room temperature with each MoAb. Red blood cells were lysed by incubation with Excellyse I (EXBIO, Praha, CZ), followed by incubation with distilled water. Samples were washed and resuspended in phosphate buffered saline (PBS). All reagents were used according to the manufacturer's instructions. All samples were processed within 48 h of collection.
Downstream tips
Immediately after preparation, samples were analyzed on a FACSCalibur flow cytometer using CellQuest™ Pro software (BD Biosciences, San Diego, CA, USA). About 100,000 events per sample were obtained. Infinicity™ Flow Cytometry version 1.7 software (Cytognos, SL, ES) was used for data analysis.

Manufacturer protocol Publication protocol 1 Relevant paper

PE anti-human CD52 Antibody

BioLegend

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) as reported

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) as reported

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) as reported

Manufacturer protocol Publication protocol 1 Relevant paper

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