I am curious if anyone has been successful using a vacuum manifold to wash FACS samples instead of centrifugation to save lots of time if stain samples in a 96 well millipore filter plate? We could do this but all I could find online is that removing all liquid from the cells may stress them too much if not fixing until after surface staining but that was only 1 person's experience. Hope other people have tried this and have a few tips. I would like to stain multiple surface markers on various human immune cells, wash and then fix with PFA but with a full plate I definitely dont want to use tubes and the alternative is a 96 well v bottomed plate but I'm always a bit cautious of flicking the plate upside down after spinning. Thanks for any tips/advice!
While I haven't tried the vacuum method, I do have some experience using the 96 V-shape plate method. I have used it on unfixed ESC-derived cells, satellite cells and total bone marrow cells without much problems. Initially, I was very skeptical and thought that most of my cells would just fell off the plate when I turned it upside down, but it actually works pretty well. I do use a higher spinning speed though: 2500 rpm for 3 min (I normally use 1200 rpm for tubes). However, since it is kind of difficult to get rid of all liquids using this method, I wash the cells at least twice (washing once is usually sufficient for my cells if I use tubes). Also, if you are going to do intracellular staining, cells become very light after the permeabilization step and I will spin them at a even higher speed. But it still works.
I have been using polystyrene V-bottom plates to successfully stain PBMC, both unfixed and fixed (a variety of ways, including methanol). I spin for 2-3mins at 350xg (unfixed) or 450xg (fixed). I feel that I lose fewer cells than using facs tubes. To avoid spill-over, flick once and DO NOT blot the surface of the plate. Also, use a plate with 300ul size wells, washing with
Deanna,
I basically agree with all, especially with Fernando. All I like to add is that 1) PBL (fixed or unfixed) are easy to resuspend in PS-96V plates by gently vortexting (you can actually watch from below the pellet disappear after vortexing), when spun normally ~1200 rpm/5 min not as fast as Sunny recommended. It's definitively not necessary to spin fast (you may spin ~1500 for fixed/permed cells however). 2) Cells in the much more expensive Filter Plates are probably also much harder to resuspend, and likely require thorough pipetting. It will offset much of the time savings when multiple staining/washing steps are required.
I tried a number of 96-well filter plates with varying pore sizes for this application. With smaller pore sizes (that exclude cells), the filter gets clogged quickly when vacuuming. With larger pore sizes, the cells may go through the filter (and it still gets clogged). In both cases, I ended up with fragments of the filter in my cell samples after removing, although it is not a huge deal bc they can be excluded by FSC/SSC. It's a good idea for saving time, but not technically feasible. I spin 96-well round bottoms @ 1600 rpm x 3 min and flick.
Thank you all for your responses! It does seem v bottomed plates would work fine and maybe our multichannel aspirator could work or else I will flick once and not blot as most mentioned. I do like using tubes if small # of samples as then bigger volume and only 1 wash but not too bad if can flick plate versus aspirate. I didn't realize a quick vortex would resuspend enough just for washing so that would definitely save time and pipette tips.
For the v bottomed plates what is the maximum number of cells I wonder that would fit in the bottom? I have also now tried a PCR plate since the wells are nice and pointy and I do get nice pellets in there as well but prettty sure PCR plates are more expensive than a v bottomed plate. Thanks again
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