Summary Document
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Hello
I received many helpful responses to my question regarding small sample size sorting. Thank you so much to all of you who responded.
I really appreciated hearing what you do and do not do to make these type of sorts optimal.
I have collated the responses in the text below.
Thanks again,
Joanna
Flow Cytometry Facility
AI 0129 EPFL
SV-SG Lausanne
Bâtiment AI Switzerland
+41 21 69 39 547
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QUESTION About:
Sorting with a small sample size
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REPLIES:
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Of course you'll NEVER get 100% back in any real world.
Almost every time there is a coincident or even nearby cell, it will be unwanted. You could use the abort-save mode to collect aborted cells and then re-sort (over and over!)
I just move the sample support arm over and let the sheath backflush into the tube for a few seconds and then restart. No knob turning.
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25000?? And your costumer wants a population of 1%?? Costumers are crazy!
I suggest you to dilute as much as you can your sample and run it very very slowly, because you'll have very small amount of target cells (250 cells). If you run your sample quite fast, probably you'll lose all your cells because they'll be rejected in "contaminated" drops.
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I used to sort these kind of samples all the time. I'd sort at low pressure (12psi) and never have less than 0.5ml for a sort sample. You're event rate is going to be pretty low, 100-500 per second or even lower. That's fine, that should help increase your efficiency and purity. The hard part is the set up, especially if you're dealing with overlapping fluorochromes, and often these folks don't have enough cells to spare for proper compensation controls. So, the more you can utilize fluorochrome combinations like FITC and APC, to avoid compensation issues, the less cells you'll spend on set up and be able to get everything back. The cells I sorted were embryonic stem cells, so we used a 90 um tip as well.
ps you can also use the BD comp beads for compensation controls if you really need to compensate...
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The best thing you can do is to tell the customer to bring more cells.... and you are correct in the only thing you can do is back flush then vortex the tube to get any cells from the sides of the tubes...
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Joanna, I usually sort very small populations and what I typically do is start from a very large number of cells, then preselect using a magnetic column and then I take the enriched sample to the sorter..... If your costumer has this option this might be a good way to start.
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We backflush our sample line all the time too. If there are very few cells as in the sample you speak of, I'll probably do it twice. Once the tube runs dry, any sample in the sample line is stuck in the sample line. I might even add 100ul of sheath fluid from your sample stream, and run that to push the remaining cells out of the sample line!
Also, another trick I learned from BD's FACSort manual, is to coat your FACS tubes with 4% BSA before putting your small sample in the tube, to prevent cells from sticking permanently to the plastic. This really works. The protocol says to incubate the FACS tubes with a 4% BSA solution made up in PBS for 30 minutes before adding cells. Simply pour out the BSA solution (recycle and reuse for other tubes if you want) and use for your cells.
I've also gone so far as to suggest to the person handling the cells initially to pre-coat pipettes or pipette tips with BSA as well, before using them with the cells, as the cells will happily stick to any plastic along the way, increasing the cell loss before they even get them to the sorter!
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I often do something similar; just take the sample tube off the stage (still on "run") while keeping the sample line in the tube to collect the back flushed drops. I find this can often help in getting that very last cell sorted; this is especially true if you have the inline filter on the end of the sample line. In this case I often repeat the process 1 or 2 more times. The only caveat is that your cells are now sitting in "pure" sheath fluid and some may not be very happy with that. Although I have found that human lymphocytes do not appear to mind.
I also sort slowly in these cases; helps to keep coincident events and therefore abort rate down.
Before back flushing I also usually "wash out" the sample tube with a few tens of microlitres of whatever buffer/medium the investigator uses to suspend his cells.
Also advise your investigator to keep a healthy amount of serum in his buffers/medium, it will help stop cell sticking to the sample tube.
I have also heard it said that using polypropylene rather than polystyrene sample tubes will reduce the number of cells lost through sticking to the sample tube.
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