All the macrophages are dead - (Oct/17/2008 )
Hello
When I stimulate my macrophages (primary cells/BM) with LPS/IFN-g (10/10 ng/ml) during two days, almost ALL cells die, confirmed by counting with Trypan Blue and propidium iodide. So my question is if my stimulation is too strong or maybe the way I detach the cells from the well plastic, I use versene and incubate the cells for 30 minutes, and then I use a pipette to collect the cell suspension, the cells do not detach by themself even with Versene, I need to use the pipette also, but with very little force.
Any ideas?
Thanks
When I stimulate my macrophages (primary cells/BM) with LPS/IFN-g (10/10 ng/ml) during two days, almost ALL cells die, confirmed by counting with Trypan Blue and propidium iodide. So my question is if my stimulation is too strong or maybe the way I detach the cells from the well plastic, I use versene and incubate the cells for 30 minutes, and then I use a pipette to collect the cell suspension, the cells do not detach by themself even with Versene, I need to use the pipette also, but with very little force.
Any ideas?
Thanks
hi
normally cells lift up and float in the medium when they die, no need to use propidium iodide or trypan blue for that case.
are your cells too confluent?.......maybe if the confluency is 100% after 2 days (post treatment with your chemicals of interest) they die by over population !
why do you use pipette to detach cells? why not Trypsin or scrpaers?...sorry I haven't worked with macrophages so I'm not sure if it's a right method you are doing.
When I stimulate my macrophages (primary cells/BM) with LPS/IFN-g (10/10 ng/ml) during two days, almost ALL cells die, confirmed by counting with Trypan Blue and propidium iodide. So my question is if my stimulation is too strong or maybe the way I detach the cells from the well plastic, I use versene and incubate the cells for 30 minutes, and then I use a pipette to collect the cell suspension, the cells do not detach by themself even with Versene, I need to use the pipette also, but with very little force.
Any ideas?
Thanks
hi
normally cells lift up and float in the medium when they die, no need to use propidium iodide or trypan blue for that case.
are your cells too confluent?.......maybe if the confluency is 100% after 2 days (post treatment with your chemicals of interest) they die by over population !
why do you use pipette to detach cells? why not Trypsin or scrpaers?...sorry I haven't worked with macrophages so I'm not sure if it's a right method you are doing.
Yes that I know, there is some floating cells (10-20%) but they are washed away. So the living cells are still alive, the cells are not confluent, it is 1E5 cells/ml in a 16 well plate which is ok. I can see that the cells have a lot of vesicles in the cytoplasm which is normal due to the NO production and the stimulation (LPS/IFNg). I pipette the solution up and down in the well so that the cells releases from the bottom. I cannot use Trypsin because I want to detect the receptors by FACS.
Hi,
If the cells look good before you try to collect them than the problem is with the way you are collecting them.
try to wash the cells 2-3 times with HBSS ca/mg Free (it's better than PBS).
Incubate the cells with Versane for 15-30min @ 37c and than take the cells off using a blue tip (1ml). DO NOT scrape the cells !!!
If the cells look good before you try to collect them than the problem is with the way you are collecting them.
try to wash the cells 2-3 times with HBSS ca/mg Free (it's better than PBS).
Incubate the cells with Versane for 15-30min @ 37c and than take the cells off using a blue tip (1ml). DO NOT scrape the cells !!!
Hey!
Thanks for the quick respond!
That is exactly how I do, I use Versane for 30 minutes in a incubater (37 degrees) and then get them of by using a blue tip. I need to flush the solution many times go get the cells to release from the plate. I think that maybe Im using to high IFN-g concentration, in the datesheet the ED50 is ~1 ng/ml, im using 10 ng/ml but that is what many people are doing, I mean the receptors become saturated anyway.
I will also try to use a shaker in the incubater (during 30 min) maybe then they survive... I maybe forgot to mention that I stimulate them during 48 hours, this is due to that I want to have high MHC class II expression.
The washes with the HBSS are crucial (from my experience, i know it sounds ridiculous....).
By that way, in my hands MHC-II is a bit tricky to detect
I've never used Versane before, we incubate the cells in PBS (but you can use HBSS, I'm sure) on ice for 10 min. Then pipet up and down to get them off. Pretty simple, although I'm not a 100% sure how it effects MHC class II sincen nobody in my lab has ever looked at that.
PBS won't effect it. Are you using too strong a concentration of versene/EDTA? You shouldn't need it.
Resort to scraping if you have to - just be gentle, scrape with media present (don't tip/tilt media away) and use the rubber/soft plastic tip scrapers - you should get very little death.
Resort to scraping if you have to - just be gentle, scrape with media present (don't tip/tilt media away) and use the rubber/soft plastic tip scrapers - you should get very little death.
I use 1x Versene and it has been used before, in other articles, it might be that they become overstimulated, but they are not floating, that is the weird thing.