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Flow cytometry of neutrophils

PostPosted: February 11th, 2015, 1:24 pm
by Sophs012
Hi,



I'm currently doing flow cytometry on neutrophil apoptosis. I'm seemingly having an issue with the number of events going through the flow, from 150 - ~3000 when I intially load 300,000 neutrophils at the start (I'm aiming for 10,000 events). I can't see why I have this issue as other users in the lab haven't had this problem



Any thoughts?



Sophie

Re: Flow cytometry of neutrophils

PostPosted: February 11th, 2015, 7:55 pm
by BioWizard
I know this sounds silly, but the first thing I would check is whether you're getting your input cell counts right. Have someone from the lab watch you count and go over your math. Or even better, have them count independently and make sure you're getting roughly the same numbers. If you are, then maybe we should look into the details if your staining, and what steps you're following precisely.

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 4:33 am
by Sophs012
Hi,

that's quite useful. But assuming the inital maths is right, where could I lose cells?

My protocol is:

1) lift cells from wells (checking under the microsocpe that they are lifted) and spinning down at 8000 RPM for 5 mins
2) remove supernatenent, wash with a bit of media (I've established this as a potential problem area, but do usually take care not to disrupt the pellet) and resuspend the pellet into 100microlitres of media (assuming 1 million cells are still there)
3) aliqout 30 microlitres (300,000 cells) into plate or tubes and add HBSS (100 microlitres for plate and 1 mll for tubes) and spin the other 700,000 cells down at 8000 rpm for 5 mins for protein extraction
- of note I usually use the tubes for the staining, but tried with the plate to see if less volume will concentrate the cells up - didn't work very well. Also when I spin down the cells for protien, there is a pellet still, though smaller than the first one, thus washing doesn't seem to be too troublesome
4)spin down plate/tubes at 1500 RPM for 5 mins and remove supernatent - I was wondering if this was high enough to pellet the cells so that when the supernatent is removed the cells aren't lost with it
5) add 100 microlitres of HBSS and 1 microlitre of annexin V and leave in the fridge for 15 mins
6) add 900 microlitres of HBSS to tubes plus 1 microlitre of PI/dilute PI 1 in 10 and add 1microlitre to plate and leave in dark cupboard at RT for 2 mins
7) centrifuge at 1500 rpm for 5 mins, remove supernatent and resuspend in 600 microlitres of HBSS (tubes) or 200 microlitres (plate)
8) read

I have a couple of ideas where there may be loss but I'm not sure if they have any weight - but then I suppose that's science, its usually something very simple and tiny!

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 7:13 am
by BioWizard
First thought, 8000 RPM strikes me as too high for pelleting cells, especially when a later step uses 1500 RPM (suggesting that 1500 would be sufficient to bring them down). How many g's does 8000 RPM translate to on your centrifuge? Usually around 80 g's is enough for most of the cells that I work with.

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 7:45 am
by Sophs012
Hi,

On our centrifuge, 8000RPM is about 6093 g - but I am using the protocol established by the lab on this.

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 7:51 am
by BioWizard
Why would you need that much g to just pellet down cells? I've read that 250 g is enough. Some protocols go to 800. probably for gradient stuff. But 6000? Using too high g will burst cells and cause cell death/loss.

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 8:03 am
by Sophs012
We use it for protein/RNA extraction, would you suggest lower for the initial spin?

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 8:07 am
by BioWizard
Sophs012 » 12 Feb 2015 08:03 am wrote:We use it for protein/RNA extraction, would you suggest lower for the initial spin?


Yes of course. For subcellular components you need to go high to bring them down cause they're much smaller/lighter (such as when you're trying to clear a protein/RNA prep from debris). But whole cells would burst if you go too high. Imagine pushing down with high force on a bunch of water balloons.

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 8:08 am
by Sophs012
Okay, I think that may be the problem! thank you

Re: Flow cytometry of neutrophils

PostPosted: February 12th, 2015, 8:12 am
by BioWizard
Sophs012 » 12 Feb 2015 08:08 am wrote:Okay, I think that may be the problem! thank you


No problem, good luck. Let us know if that solves it or if we need to keep thinking about it. Though I suspect that might have been it.