Scott, I previously worked in a bone marrow transplant lab, and I don't think that you would have much problem freezing these cells. Most stem cell labs freeze with a protocol similar to this: After collection, spin the cells down and resuspend the pellet in a minimal amount of the plasma you took off. Determine the total volume that you'd like to freeze in. (Most facilities do this based on the WBC concentration, and anywhere from 1 to 10x10^8 WBC's / mL is fairly common.) 10% of the total freeze volume should be DMSO. The rest should be made up with the plasma you spun off. For example, if you had 10mLs cells, and determined that you wanted to freeze in a total volume of 50mLs, you would have: 10 mLs cells 5 mLs DMSO 35 mLs plasma SLOWLY combine the DMSO and the plasma, and then chill this on ice until it's cold to the touch, the colder the better. (An exothermic reaction takes place when you add it, and it can get pretty hot, hence the precaution of cooling it first so that you don't "shock" the plasma.) Again slowly, add this cooled "freeze media" to the cells. At this point, my lab would use controlled-rate freezers to freeze the product, but many labs simply drop them straight into the liquid Nitrogen, and don't report many problems. (I don't have any experience with this technique, however.) I frequently used this process on a smaller scale (like you are proposing) and it worked quite well. But you must resuspend the cells in media with 10% DMSO, or the recovery upon thawing is atrocious. To thaw, you simply run the bag under warm water (or hold the vial in your warm hand). It is important, however, to wash the cells immediately upon thawing, as the DMSO is toxic to them if they stay in it. Frozen samples have demonstrated good recovery for 10 to 15 years. Hope this helps! Nathan D. Foushee, MT (ASCP) Product Specialist - Flow Cytometry Give us a call- Let's MultiPlex! www.QuantumPlex.com Bangs Laboratories / FCSC 9025 Technology Drive Fishers, IN 46038-2886 800.387.0672 317.570.7020 fax 317.570.7034 nathan@bangslabs.com www.bangslabs.com www.fcstd.com ><SManetz@Therimmune.com> wrote: > >The lab I do work for has studies about to get underway in rats and >monkeys that have blood samples (into EDTA coated tubes) being taken at >multiple time points. We have always analyzed fresh blood, but these >studies are smaller and it would be better if we could wait until the >end to analyze everything instead of wasting time. I am interested in >spinning down the blood, removing the plasma, and then freezing the >remaining cells for analysis of PBMC subsets. My questions are: > >1. We always used whole blood and lysed rbcs after staining, but can you >take a blood sample, spin it, take off the plasma, resuspend the pellet, >and still get the same values as if you had stained whole blood? >2. Should I just freeze the intact cell pellet after removing the plasma >or resuspend the pellet in media and then freeze? >3. If I need to resuspend in media, does it need to contain DMSO or be a >freezing media? >4. How long are the cells good for once frozen? > >Any help would be greatly appreciated. > >Scott Manetz
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:40 EST