PE Mouse anti-Human CD52

Protocol tips

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.

Publication protocol

"The panel with the four MoAbs under study was distributed using the following fluorescences: CD123 FITC (clone 7G3), CD52 PE (clone 4C8), CD43 APC (clone 1G10) and CD200 PerCP-Cy5.5 (clone MRC OX-104). All MoAbs were purchased from BD Biosciences (San Diego, USA). 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.7

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. For the gating strategy, debris were removed, based on forward-scatter (FSC) and side-scatter (SSC) distribution. Neoplastic cells were initially identified by positivity of the CD19 and CD20 and then in accordance with the expression of other panel markers for MBCN. The mean fluorescence intensities (MFIs) of neoplastic cells were recorded in arbitrary units from 0 to 104."

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