Patient blood was collected in sodium citrate cell preparation tubes (BD Biosciences). Tubes were centrifuged at room temperature for 30 min at 1650 x g without brake. The mononuclear cells and plasma layer were then transferred to a 50 mL conical tube and centrifuged at room temperature for 10 min at 256 x g. Supernatant was removed without disturbing the cell pellet. Cells were resuspended in PBS, and all cells of the same subject were combined into one 15-mL conical tube. Cells were centrifuged as above. Supernatant was removed without disturbing the cell pellet, and cells were resuspended in 5mL of ACK lysing buffer (Thermo Fisher) for 5 min. Cells were washed with PBS, counted, washed again with PBS, resuspended in 10% dimethylsulfoxide in FBS at 106 cells per mL and aliquoted into cryovials. Cryovials were transferred to a freezing container (Daigger Scientific, Mr. Frosty) and placed in a −80°C freezer overnight before being transferred to liquid nitrogen for storage.
Sodium citrate cell preparation tubes
Manufactured by BD
Sodium citrate cell preparation tubes are laboratory equipment used to collect and store blood samples. They contain sodium citrate, an anticoagulant, which prevents the blood from clotting during the collection and preparation process.
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Lab products found in correlation
2 protocols using sodium citrate cell preparation tubes
1
Isolation and Cryopreservation of Peripheral Blood Mononuclear Cells
2
Isolation and Characterization of Bovine PBMC
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Well-mixed blood from the remaining K2 EDTA tubes was transferred to sodium citrate cell preparation tubes (BD Biosciences). Cell preparation tubes were centrifuged at 1,800 × g for 30 min at 20°C. After centrifugation, platelet-rich plasma was discarded without disrupting the density-gradient separating PBMC. The PBMC were collected, washed, and pelleted at 300 × g for 10 min at 20°C in autoMACS Rinsing Solution (PBS, pH 7.2, and 2 mM EDTA; Miltenyi Biotec). During a second wash, red cell contaminants were lysed via osmotic shock using distilled water, vortexed, and immediately diluted with autoMACS Rinsing Solution, followed by centrifugation at 300 × g for 10 min at 20°C. During the third and final wash, PBMC were then pelleted at 300 × g for 10 min at 20°C and the supernatant was discarded. Before the study, cell preparation tubes were validated for isolation of PBMC in bovine blood via university veterinary pathologist and differential staining. Cell counting and viability of the isolated PBMC were determined using trypan blue and a hemocytometer. The viability of PBMC collected from multiparous and primiparous cows was not different, and the mean viability was 90% live cells.
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