CD52 Monoclonal Antibody (CF1D12), PE

Protocol tips

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

For immunophenotyping, fresh BM aspirates were obtained as a part of the clinical routine. The BM aspirates were separated into 100 μL aliquots. To detect malignant B cells, immunophenotyping was conducted with antibodies against k light chain (PE, R0436, Dako Cytomation), λ light chain (FITC, F0435, Dako Cytomation), CD19 (PE‐Cy7, HIB19, BD), and CD45 (APC‐Cy7, J33, BD). For PC immunophenotyping, an eight‐parameter flow cytometry analysis was performed using cytoplasmic anti‐κ, cytoplasmic anti‐λ, CD19, CD20, CD22, CD27, CD30, CD38, CD45, CD52, CD56, CD81, and SLAMF7 antibodies (Table 1). 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).

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