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Found 3 matching solutions for this experiment
| Upstream tips |
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For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C. |
All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies). |
| Protocol tips |
| For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C. |
| Downstream tips |
| All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies). |
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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. |
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). |
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100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence stain |
The cells were acquired in a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA) using the FACSDiva software (Becton Dickinson). A minimum of 100,000 events were acquired. For data analysis the Infinicyt software (Cytognos SL, Salamanca, Spain) was used. |
| Protocol tips |
| 100 μL of EDTA‐anticoagulated pleural fluid samples were immunophenotyped using six combinations of monoclonal antibodies and a direct immunofluorescence stain |
| Downstream tips |
| The cells were acquired in a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA) using the FACSDiva software (Becton Dickinson). A minimum of 100,000 events were acquired. For data analysis the Infinicyt software (Cytognos SL, Salamanca, Spain) was used. |
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