I am differentiating PBMCs to iDC and currently leave the monocytes for 6 days with the cytokines, however after day 2 of 6 I notice dead cells (some monocytes and some DC). Do these cells have an effect on the differentiation of monocytes to iDC and how does one get rid of the dead cells and debris (what centrifugation speed and time) ? I currently use 200g 10 min but it brings down dead cells as well. I change half the medium on day 2 and day 4.
You should have a threshold for your CD14+ as a quality control measure. Are you getting the appropriate % of CD14+ cell?
I would also suggest using fresh media from a company -- make sure the media is used within the first week of the date you receive it. Old media ( more than 1 month old) can sometimes cause deleterious effects on your cell culture. Since you are trying a new protocol, I would make sure that everything is fresh and new. That would help you establish qc threshold.
Its hard to comment on the specifics of the protocol since it is not presented above.
I have noticed it as well when I generate my iDCs from positively isolated CD14 from PBMCs. When I looked at the differentiation of monocytes daily, the biggest cell death occured at day 1, when (for some strange reasons) almost half of my monocytes died off and the rest was nicely differentiating towards iDCs. Some say that it is the case when isolated monocytes are cultured without any cytokines. However I do see it with cytokines as well. I found also that it is could be donor dependent. At day 6 when I harvest my iDCs, sometimes I have relatively big population of dead/dying cells. Then other times (different donor) it is nice and fairly clean population of DCs. I started using Dead Cell Removal Kit (Annexin V-bound beads,Miltenyi) which nicely cleans up my cell population from dead cells. it is also straight forward protocol, which you can find on Miltenyi website.
you could also use Histopaque, where you layer your sample on it and centrifuge it without brakes at 400g for 30min. Aspirate the upper layer and collect the layer just above the histopaque. wash the collected cells with PBS or media.
Hope this helps!
Good luck!
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