Hi everyone,
While doing FACS from human blood cells, I felt cells to be very sticky and hence they form clump which is very difficult to re-suspend in PBS.
PBS, I use is calcium and magnesium free.
They even firmly stick to pipette tips that causes loss of cells.
Please advise some solution.
I guess it depends on the cell types you're working with. Some people add EDTA to their FACS buffer..
What cell population from blood are you looking at? I have run FACS on DCs isolated by percoll gradient after 24hr plate incubation, and used PBS+EDTA to detach them from the plate surface rather than trypsin to preserve surface receptor expression and found this worked well and prevented clumps. You can then neutralize this with PBS + calcium and magnesium, if this would not be detrimental to your protocol.
Good luck!
Doing a red blood cell lysis step on your sample (unless you want to look at these!) will help to get rid of RBC and any debris - solutions are widely available for this.
Also make sure you do wash steps if using freshly isolated PBMCs - PBS for this is fine - and filter before staining. For actual staining and/or running, i have used 5mM EDTA and 4% FCS and havnt had any problems. You should also filter prior to running to get rid of clumps. Hope this helps!
Dear Pooja, if you have a lot of dying-dead cells in your samples adding DNAse will also stop the clumping of cells
When working with cultured PBMC, these tend to form clumps, but easily can be treated with EDTA as indicated above. In flow cytometric analysis, you could try to discriminate singlets by the strategy FS-A vs FS-H.
2mM EDTA with 1%FCS in PBS will help. If is possible, lyse the RBC before will definitely help a lot.
Try adding DNAse to your sample and incubate at RT for 15 min. It will help in dissociation of clumps.
Thanks to all for your valuable suggestion.
I used to collect cells after RBC lysis step using RBC lysis buffer and store them at -200C until further use.
Just gentle dissociation mechanical- even sterile scalpel-
but never ever ever ever ever.....never use any enzyme that some less than well read- for the want of a suitable word on a public forum-might recommend-
no DNASe, hyaluronidase, not even trypsin.
Even 0.01 trypsin affects the cell surface (Garth Nicolson- 1970).
Enzyme does the work, release surface material- never ever recovers fully- as a thank 'you' some it remains adsorbed- gets desorbed and will 'infect' fresh/virgin cells and act where you may not wish the enzyme to act.
Surely that is not what you to do 'knowingly'- in ignorrance.... well...
This tragedy of ruining perfectly good cells must be stopped.
There are papers : 1973, 1974: Sachtleben et al. 1973. Vox; Mehrishi et al. Behring Inst. 55, 1974; Seiler, Sedlacek, Behring Inst Mitt 55: 1974; Hodges. J Cell Sci, 1973.If you want FITC, EM pictures other data- see TRANSFUSION 2013: 2667-suppl material or write to me.
Hi
For FACS sorting you should use cold PBS containing BSA 1% and EDTA 1-5 mM.
Best
Hi everyone,
Recently, I have used 25mM HEPES buffer and 5mM EDTA in 1XPBS for antibody dilution and washing. Although, clump formation was less but it was present.
Besides, I would also like to know that do the presence of HEPES buffer and EDTA interfere with the result while making antibody dilution buffer.
You are quite right to suspect/question the use of EDTA- Using EDTA though suggested by the great experts sadly/totally unaware that it tends to destablise the membrane with Ca and Mg ions missing. HEPES should be fine.
Hi Pooja,
I'm not entirely sure if this has been addressed as of yet, but if you are still getting clumping with the use of HEPES and EDTA, I'd recommend purchasing 70 um nylon mesh. It comes in a large sheet and you can cut it to fit your plate and/or tube rack. Once you've cut it to size, apply your stained/fixed sample within the pipette tip firmly against the mesh pressed over the opening of the FACS tube, and depress the pipette firmly to dispel your sample quickly through the mesh. This mechanical dissociation should really help with the clumping.
As an aside, I use 2 mM EDTA in my FACS buffer after my staining is complete, and that works well for me. EDTA acts as a chelator, and reduces cell clumping that is cation-dependent. DNase is used to eliminate the DNA freed by the dead cells in your sample, and is recommended should you find/expect many dead cells within your sample.
Are you running your sample in the plate or by HTS? I find that clumping occurs more rapidly when using HTS.
Best of luck to you!
Hello Pooja ,
you can use ice cold HEPES+2% FBS or PBS + 10% FBS throughout the experiment . You need to keep your cells in ice. Most importantly, you need to vortex your appendorf before acquiring in flow cytometry.
Best regards,
Babu
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