Whole blood samples (with heparin) were centrifuged at 1850 rpm for 15 min with breaks off. Subsequently, the upper fraction (plasma) was collected and stored at −80°C. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE). 35 mL of RPMI 1640 medium (RPMI, Omega Scientific) diluted blood was slowly layered on top of 15 mL Ficoll-Paque PLUS. Samples were spinned at 1850 rpm for 25 min with breaks off. Then, PBMC layers were aspirated and two PBMC layers per donor were combined in a new tube together with RPMI. Samples were spinned at 1850 rpm for 10 min with a low break. Cell pellets of the same donors were combined and washed with RPMI and spinned at 1850 rpm for 10 min with breaks off. Finally, PBMCs were counted using trypan blue and a hemocytometer and, after another spin, resuspended in FBS (Gemini) containing 10% DMSO (Sigma-Aldrich) and stored in Mr. Frosty cell freezing container overnight at −80°C. The next day, samples were stored at liquid nitrogen until further use.
Ficoll paque plus
Manufactured by Omega Scientific
Ficoll-Paque PLUS is a density gradient medium used for the isolation and purification of cells, such as lymphocytes and other mononuclear cells, from whole blood or other cell suspensions. It facilitates the separation of different cell types based on their density differences.
Automatically generated - may contain errors
2 protocols using ficoll paque plus
1
PBMC Isolation and Cryopreservation
2
PBMC Isolation from Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole blood samples (with heparin) were centrifuged at 1850 rpm for 15 min with breaks off. Subsequently, the upper fraction (plasma) was collected and stored at −80°C. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (Amersham Biosciences). 35 mL of RPMI 1640 medium (RPMI, Omega Scientific) diluted blood was slowly layered on top of 15 mL Ficoll-Paque PLUS. Samples were spinned at 1850 rpm for 25 min with breaks off. Then, PBMC layers were aspirated and two PBMC layers per donor were combined in a new tube together with RPMI. Samples were spinned at 1850 rpm for 10 min with a low break. Cell pellets of the same donors were combined and washed with RPMI and spinned at 1850 rpm for 10 min with breaks off. Finally, PBMCs were counted using trypan blue and a hemocytometer and, after another spin, resuspended in FBS (Gemini) containing 10% DMSO (Sigma-Aldrich) and stored in Mr. Frosty cell freezing container overnight at −80°C. The next day, samples were stored at liquid nitrogen until further use.
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