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Found 3 matching solutions for this experiment
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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). |
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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) |
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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) |
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>20 × 105 disaggregated sphere cells were stained with a stem cell‐specific antibody panel and analysed on a BDTM LSR II flow cytometer. To a volume of 100 μL cell suspension, 5 μL antibody mix was added, mixed and incubated for 20 minutes at 4°C in the dark and washed twice with 250 μL DPBS or FACS buffer. |
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>20 × 105 disaggregated sphere cells were stained with a stem cell‐specific antibody panel and analysed on a BDTM LSR II flow cytometer. To a volume of 100 μL cell suspension, 5 μL antibody mix was added, mixed and incubated for 20 minutes at 4°C in the dark and washed twice with 250 μL DPBS or FACS buffer. |
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