Hi all,
I have been scouring the internet for a protocol for defrozen PBMC fixation and embedding.
Currently I am defreezing PBMCs with an in-house protocol which gives me ~95% viability. After pelleting and washing (PBS no BSA) I fix my sample with 4% PFA overnight (~16h) followed by 2X wash with PBS (no BSA) and spin down @ 800g for 8min in 15mL Falcon tubes.
The issue I am encountering is massive cell loss and usually end up with barely 5% of my initial cell count.
Considering that I use standard histology protocols I have a hard time understanding what the issue is. Of note, cells maintain their morphology after fixation and clearly the loss intervenes at the washing stage.
Would using PBS with 0.2% BSA for the post-fixation help me pellet better and keep more cells?
Overall, if anyone has a good protocol to recommend for FFPE preparation of PBMCs I will gladly take your advice.
Thanks in advance :)
Hey Tiffany, it's more variable than a consistent 95% really, it's more consistently around 75-80%.
For 5 mio cells in 0.5mL with 10% DMSO, we dilute the DMSO with cRPMI up to 32mL minimum before a first spin. I try to keep everything at RT and limit massive heat shocks (transfer in dry ice after liquid nitro and immediately into the 37° bain marie)
I don't understand what your goal is with fixing and embedding those PBMC cells. FFPE is used for sectioning tissues. Whaf do you want to do with the embedded PBMCs? FFPE method is not suitable for defrozen tissues or cells. During freezing, ice crystals were made, if you didn't use adequate media for preventing the formation of ice crystals. Unfortunately, you cannot get good morphology of PBMC or any kind of tissues or cells after freezing. Once your tissue is frozen, you only use cryostat for sectioning. But I've never heard of freezing, fixing and paraffin-embedding of PBMCs.
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