Hello everyone,
I need to know if the MDSCs cells can be isolated by magnetic beads, without using the flow cytometry to sort them, I found in an article a comparison between MACS and FACS techniques, but I didn't understand well if it's a necessary to follow it by flow cytometry or not.
Thank you.
I don't have experience with MDSCs but I do with MACS and FACS. Using MACS you will have a very enriched but not 100%. On the other hand, using FACS you may achieve >95% (close to 100%). If you use MACS you will need to access cell purity through flow cytometry. So, basically the method will depend on the purity you need and the viability of your cell after the separation protocol.
I found this post on SC sorting (MACS vs FACS). You should take a look.
http://stemcellassays.com/2013/01/facs-vs-macs/
If you need to know the purity of the enriched fraction (as the preparation will give varying results you should always check this) you will need a method to evaluate it and this is usually done with flow cytometry. One could probably also stain a sample aliquot and count them on a fluorescent microscope but this will be more time consuming.
Thanks Mr Andre, for your explanations, and for the post, they were helpful.
Christa G. Walther, it will be a waste of time. So, the best methode is to sort by flow cytometrie?
I have used both FACS and MACS sort for hematopoietic and non-hematopoietic populations. Yet, I would recommend that you do Flow instead of MACS. The accuracy/purity is a bit better in FACS. Besides, when you use multiple antibodies, FACS is strongly recommended. Best of Luck.
Depending on expected frequency of population of interest, MACS is usually used to enrich the population of interest from huge number of starting cells (for example human CD34+ fraction are only around 1% of healthy donors PBMCs, so positive MACS will give you appr.100fold primary enrichment), then it will be easier and faster to sort rare population of interest (usually by multicolor flow cytometry). But remember that some remaining magnetic beads (on cells that were not removed by magnet) can give you autofluorescence in 488nm laser/green channel, so need to have unstained control from after-MACS fraction to separate this unwanted cells from those really positive for antibodies with green fluorescence (FITC, Alexa Fluor 488, etc.). Also in cancer patients CD34 epitope, for example, may be modified and not very well separated by MACS in general, depending on antibodies clone used for conjugation with magnetic beads by the companies. So doing flow cytometry after MACS is a must to see the purity of your population and to see how many cells of interest remained in "throw away" fraction.
if you are using MACS I stongly recommend to enrich your population on interest by negative selection. From my experience I feel Positive selection in MACS could decrease the viability and might activate the cells due to antibody binding. So based on the assay to asses the population and deplete cells you could use MACS. Where as to asses the percentile of different populations with two or more antibodies it is alwyas better to use FACS. If you are planning to use flowcytometry to separate the cell population and use later it give better results but contamination and lower levels of other cells would be the problem even in this technique.
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