CD30-PE, HRS4, 2 mL, ASR

Protocol tips

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.	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
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).

Publication protocol

"BM aspirate specimens were collected in EDTA‐anticoagulant and processed within 24 h (h) of collection, and all experiments were performed in Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Instrument performance was checked daily by recording fluorescence intensity with calibrating beads (BD Biosciences). Reagent and antibody performance was checked by analyzing control cells (CDChex; Streck Laboratories, Omaha, NE) and peripheral blood from blood bank donors at MD Anderson Cancer Center. 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. All cases included had a substantial number of neoplastic plasma cells detected by Tube 1: CD38‐FITC; CD28‐PE; CD138‐APC; CD117‐ PE‐Cy7; CD19‐PerCP‐Cy5.5; CD56‐V450; CD45‐V500; and Tube 2: cytoKappa‐FITC; cytoLambda‐PE; CD138‐APC; CD38‐PerCP‐Cy5.5; CD20‐V450; CD45‐V500, similar to an approach described previously 19. Each case was analyzed with the above markers as part of the routine clinical work‐up where at least 100,000 total events or minimal 200 CD38+CD138+ plasma cells were acquired. For the investigated antibodies, the fluorochromes and clones used in this study were: CD30‐PE (clone HRS4), CD44‐APC (G44‐26); CD49d‐PE (9F10), CD70‐APC (Ki‐24), CD105‐PE (266) and CD184‐APC (12G5). 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). Plasma cell populations were identified by bright CD38/CD138 expression, and further redefined based on FSC‐H/SSC‐A properties. Fluorescence minus one (FMO) (CD138/CD38 without investigated antibody) was used consistently as negative controls in all cases. The results were presented as a percentage of neoplastic plasma cells with expression of each respective antigen. Mean fluorescence intensity (MFI) ratio was calculated by comparison with negative control."

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