Currently, I'm optimizing a Celltrace-CFSE assay. I want to have very distinct peaks in my flowcytometric histograms to analyze proliferation percentages. However, to receive such peaks, I need to use a slow flowrate. What is the fastest possible flowrate to use? Current speed is 66 muL/minute.The peaks are not that bad though, but I wonder if there is a more optimal speed. Thanks in advance.
Hi,
I have used BD FACS calibur model, acquired the T cells on low mode i.e. 60 micoL/Min and it gives me very good result. I have used Modfit software to analyse the pick. You can search this paper- Enhanced lesional Foxp3 expression and peripheral anergic lymphocytes indicate a role for regulatory T cells in Indian post-kala-azar dermal leishmaniasis. J Invest Dermatol. 2010 Apr;130(4):1013-22. doi: 10.1038/jid.2009.393.
Its free in Pubmed
Hi,
I have used BD FACS calibur model, acquired the T cells on low mode i.e. 60 micoL/Min and it gives me very good result. I have used Modfit software to analyse the pick. You can search this paper- Enhanced lesional Foxp3 expression and peripheral anergic lymphocytes indicate a role for regulatory T cells in Indian post-kala-azar dermal leishmaniasis. J Invest Dermatol. 2010 Apr;130(4):1013-22. doi: 10.1038/jid.2009.393.
Its free in Pubmed
Hi,
there are different points to be addressed: the no. of cells/sec is not so important. It depends on your instrument: how much cells/sec the instrument can collect, that is the limitation 1. Limitation 2: is your instrument clean and 3: do you have correct settings of PMTs and instrument setup? Hint would be the %rCV of the bright beads (should be under 4)...
Also very important: the flow rate! To get distinct peaks (beside the parameters I mentioned before) you should always use "low mode"; because the flow stream is narrow and not so wide like using "high mode". It´s a little bit like DNA-analysis: there you also just use the low mode! very important!
If you need too much time to analyse you can concentrate your cells up to that amount of cells your machine is able to deal with (recommendation: FACSCalibur ~2000 events; FACSCantoII ~ 7000 and LSRII around 12000 events per second).
And sorry, I have to correct Debanjan: FACSCalibur model, there you are acquiring 12ul/min; 60mL/sec is acquired with the high mode!
Cheers
Sorry, I forgot the Datasheet for Calibur:
https://www.bdbiosciences.com/documents/FACSCalibur_FlowCytometry_TechSpec.pdf
Hi,
It depends also your concentration of cells in your tubes (experiments). For the acquisition , maximum of 1200 events/sec.
Regards, Marlène
Hi,
I agree with Jochen, the events/sec per se is not so important if it's within the "acquisition speed" range of the instrument, but in order to get better resolution (sharper peeks) of your populations it's better to acquire in low mode, if possible.
Cheers
Thank you for your answers, I'll take these in consideration in my next measurements!