Hi,
I received a blood bag for PBMC isolation with subsequent T-cells triage required. I received it yesterday evening and put the bag at +4C. I wonder if I can do a ficoll etc NOT the next morning but the morning after? So it will rest for 24+ hours at +4C... Is it bad for the experiment?
Thank you in advance!
Keeping blood for this much of time may cause activation of granulocytes which may appear in mononuclear fraction during Ficoll separation.
Hello Mark,
Do you have the shaker at 4oC?. I think keeping the blood overnight (12hours) is acceptable, I have never tried keeping it longer. In my case, T cell looks quite good but Monocyte does not. So please consider and try to shorten your storage duration at 4oC.
I process peripheral and cord blood regularly for lymphocyte isolation. I have found if you need to leave a unit overnight I keep it at room temp and the T cell the viability is better than at 4 degrees. I would not keep it longer than overnight.
I agree with Suzanne – Room temperature storage increases viable cell yield and reduces activation and cell death induced by 4°C storage overnight. I believe 4°C makes membrane RAFT areas more rigid - locking signaling molecules together that may have been mean to be a transient interactions. People who work with granulocytes do NOT use blood stored overnight at 4°C because that process activates the cells.
To attract T cells from whole blood, the blood should be stored in the dark overnight at room temperature. Storage in a refrigerator results in a reduction of CD4 T-cells corresponding with a shift of the CD8 / CD4 ratio. We were able to perform T-cell stimulation assays, FACS analyzes, and T-cell culture.
I saw a paper (don't have it at the moment) that diluting the blood with PBS helps reducing the granulocytes activation if you need to store the blood overnight. I have not done extensively testing, but it seems to help.
Diluting blood with Normal saline or PBS (1:1) prevents aggregation of lymphocytes which will otherwise sediment in the pellet.
Isolation of Mononuclear cells should be carried out an optimum temperature which may otherwise affect viability as well as yield. Isolation of cells is based on density gradient and density is affected by temperature.
I have a question related to the topic can i store magnetic based separated CD8 T cells overnight at 4 degrees?