I am wondering whether to isolate PBMCs or do the staining on whole human blood, what are the advantages and disadvantages and pittfalls of whole blood staining. I will stain for gamma delta T cells and IL-17 but because neutrophils can produce IL-17 I might need to use whole blood.
HI! Use an ACK lysis buffer to lyse red blood cells instead of isolating PBMC. Here is a protocol and the formulation. Try to use a fresh solution. http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/buffers/ack-lysing-buffer-1x.aspx
Hi Alberta,
It is totally possible to stain whole blood for intracellular staining. I've done that lots in the past. You need to do your extracellular stain prior lysing the red blood cells, fixing, then going ahead with your intracellular stain. It all depends on what you are looking for. If you are wanting to look at the levels in neutrophils as well as in your delta t-cells, then you will obviously need to do it in whole blood. You will also have to consider the amount of blood you have available. We used to do it in whole blood as we were doing paediatric patient samples, so would get very little sample to process, so it was better to do it in whole blood. Finally, you may want to actually do both and see if you get a difference in proportions of cells, as the process of PBMC separation may preferentially select some cells out, or kill already activated cells. This can be a problem if you are dealing with patient samples, depending on what they've got. If I were you, I would do a little pilot study to test out your whole blood method on control blood, and compare that to your PBMC method and see what degree of difference you get, and then see if your patient samples do the same. Good luck!
Ha Liesbeth,
Hieronder de verzamelde antwoorden van je oude lab:
Ch. Wolf (AIO): maar er schijnen tegenwoordige steeds betere kits voor whole cell staining te zijn (bij Miltenyi geloof ik). heb ik overigens zelf geen ervaring mee. maar neutrofielen ben je natuurlijk kwijt bij isolatie van PBMC's.
Aad:
Dat klopt, wij hebben in het verleden ook wel volbloed kleuringen gedaan (BD volbloed lysis kit), echter dan zit er na de kleuring wel een lysis stap in het protocol die ook fixeert, en daarna flink wat wasstappen. Dan ben je van de ery’s en bloedplaatjes af die de FACS verstoren en de meeste FACS apparatuur op tilt laat slaan (aantal deeltjes). Dit gaat prima voor surface stainings maar voor ICS is dat een ander verhaal, Ik weet niet wat er gebeurt als je ICS kleuringen verricht met al deze verontreinigingen erbij?
Dus je zit nu aan de Zwarte Zee? Eind weg zeg, maar gelukkig ook een eind van IS af. Vakantieuitstapjes naar Georgië? Wintersporten in Sochi?
Ha Ruurd,
Leuk om weer wat van het oude lab te horen en van jou. Ik E-mail je wel via mijn yahoo-adres.
About ICC on whole blood:
There are several protocols out there when you google this subject but I wonder the experience from researchers themselves and also I cannot find publications in which they use this method. Please sent me the references specifically if they concern IL-17A.
Liesbeth.
Maybe this paper:
http://link.springer.com/article/10.1007/s00296-011-1984-x/fulltext.html
Note that SCPL1362 is from BDBiosciences! Not eBiosc.
Yes, take 100 ul whole blood, perform surface staining, treat the sample with golgi plug, fix it and perform intracellular staining. Finally treat with lysing solution and fix with 1% paraformaldehyde and acquire in flow cytometer or you can do fluorescence/confocal microscopy.
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