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Found 3 matching solutions for this experiment
| Upstream tips |
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Blood was centrifuged at 800 ×g for 20 minutes at room temperature to obtain platelet-rich plasma. Then, 1.5 mL supernatant was transferred into a new test tube and centrifuged at 1,500 ×g for further 20 minutes to obtain platelet-poor plasma (PPP). Afterward, 1 mL PPP was further centrifuged at 1,500 ×g for 20 minutes in a new polystyrene tube to obtain cell-free plasma. The top 500 µL of cell-free plasma was transferred into an Eppendorf tube and pelleted at 18,000 ×g for 10 minutes. The supernatant was carefully removed leaving 25 µL of MP-rich plasma at the bottom of the Eppendorf tube. MPs were suspended with gentle vortexing for 20 seconds in 1.0 mL Apo-binding buffer |
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| Protocol tips |
| Blood was centrifuged at 800 ×g for 20 minutes at room temperature to obtain platelet-rich plasma. Then, 1.5 mL supernatant was transferred into a new test tube and centrifuged at 1,500 ×g for further 20 minutes to obtain platelet-poor plasma (PPP). Afterward, 1 mL PPP was further centrifuged at 1,500 ×g for 20 minutes in a new polystyrene tube to obtain cell-free plasma. The top 500 µL of cell-free plasma was transferred into an Eppendorf tube and pelleted at 18,000 ×g for 10 minutes. The supernatant was carefully removed leaving 25 µL of MP-rich plasma at the bottom of the Eppendorf tube. MPs were suspended with gentle vortexing for 20 seconds in 1.0 mL Apo-binding buffer |
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100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. |
The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired. |
| Protocol tips |
| 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. |
| Downstream tips |
| The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired. |
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Protocol tips |
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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) as reported |
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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) as reported |
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