[watsond]

I am doing some work with human lymphocytes (or rather PBMCs) with regard to DNA repair of oxidative damage after overnight incubation. The only problem is that the overnight incubation causes varying amounts of dead or dying cells, as detected by using the annexin V and PI stains with a FACS cytometer. This is even observed in control cells that were not exposed to oxidative compounds. The number of dying cells is not constant between cultures, so I cannot consider any of my DNA repair results valid.

Is this something common with lyphocyte cultures of just 24h? Any thoughts about optimal culture conditions for lymphocytes?

I asked around my lab and found out that RPMI with 10% FBS works well with lymphocytes. But, I have read articles that increase the FBS supplementation to 15%. The cell concentration I use in the culture medium is between 0.5 and 1×10^6 cells/ml.

[biohazard]

There are always cells dying in lymphocyte culture, because a certain amount of cells have reached their maximum number of cell cycles and eventually die. Furthermore, some cells undergo apoptosis for various other reasons, and these numbers vary from donor to donor and thus cannot be standardised as such.

What I think you should do is to use the background signal from your control cultures (you need a control culture from every PBMC donor) as seen from annexin V and PI stains, and then compare this to your test cultures. You can then calculate a relative index of oxidative stress (or whatever you want to call it). For example, if your background signal is 10 and test culture has a staining intensity of 50, you’d get a stress index of 5 (I just made up that term, use whatever is the correct term, or invent a new one). Now, if your other culture has a background of 20 and the test culture a signal intensity of 80, your index would be 4 (so less oxidative damage there in relative terms). Or something like this, you probably got the point.

And what comes to culture conditions, 5% CO2, +37C and RPMI + 10% FCS should be just fine, although FCS can sometimes be mitogenic for T-cells, so e.g. 5% human AB serum is even a better option. And 0.5 to 1×10^6 cells /ml should be good as well. Hope this helps!

[watsond]

After trying out various culture parameters, I found that the following worked best.

Cell washing medium (for centrifuge): RPMI + 2% FCS
Cell culture medium : RPMI + 15% FCS
5% CO2 @ 37C.

After 24h incubation, necrotic cells were around 0.6% and late apoptotic cells 4.5%

[biohazard]

That sounds pretty good to me, too.

What I earlier said about using AB serum instead of FCS mainly applies only if you want to e.g. measure specific proliferation of T cells when stimulated by an antigen. But since AB serum is much more expensive than FCS, the latter is probably the optimal choice for your experiments.

[grdnfs20]

Im collecting human blood samples and isolating lymphocytes using Ficoll-Hypaque method. I am trying to culture the cells and treat them to various oxidation reactions. I am having trouble with the cell culture, there does not seem to be any control over the cell cultures, for instance i plate 500,000 cells per well and then treat them with various concentration of peroxide, which should induce cell death to some degree and im getting greater cell counts in the peroxide treatment than the control. does anyone have any insight as to culturing lymphocytes (also when removing the medium, is it necessary to trypsinize samples). if anyone can assist i would appreciate it, id also like to know if it is possible to get accurate cell viability measures using trypan blue and hemocytometer for counting. thanks

[biohazard]

You do not need to trypsinize lymphocyte cultures, they grow in suspension and do not attach to the bottom of the well. Some cells, such as monocytes do so, so if you use PBMCs to start your cultures, some cells may attach to the well, but they are not lymphocytes.

For viability measures, you may want to use erythrosin instead of trypan blue, it is pretty reliable in staining dead cells and leaving live ones unstained but still easy to count.

I haven’t used peroxide to induce cell death, so I don’t know how it actually works, but one possibility is that small concentrations of peroxide provoke cell proliferation by non-specifically activating them. Surely you can find a concentration high enough to actually cause cell death – just titre a long enough range of concentrations and then plot a cell viability curve?

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