pH of medium for lymphocyte

[DoctorStein]

I will work on lymphocytes count, which are derived from thymus and spleen. For the medium, the lab offers PBS and HBSS, each with several pHs. Which medium is better and which pH should I apply for this asessment? From references, I read that I can work with 7.2, the other mentions 7.4 even 8.0. Do this slight pH difference results in cell damage or else? Thanks for the reply.

[biohazard]

If I recall correctly, pH 7.2 should be optimal for lymphocytes, but they can tolerate some degree of variation(several decimals). At least my cells grow quite happily for a day or two in somewhat acidic conditions (= indicator colour yellow) that result from fast growth or me forgetting to replace the medium . pH 8.0 sounds too high, though.

Anyways, what comes to PBS or other non-growth medium solutions, go for pH 7.2, or something close to that.

I have a good textbook about this somewhere, if I manage to find it I can return to this topic with more precise values...

[MrMistery]

Albeit i have never worked with lymphocytes, but i did work with several cell lines of the most diverse, and they all used pH 7.2 medium. However, when it comes to lymphocytes, i am tempted to say that maybe 7.4 would be better, since that is the blood pH. indeed, just like biohazard said, the cells will tolerate a more acidic pH if you forget to change the medium or if the cells multiply incredibly fast(or if the incubator breaks).
Just a question: you mentioned PBS, but you can't culture cells in PBS. Are you using the PBS to prepare your culture medium? I have never encountered such a protocol, but i don't see why not. If you use the PBS as we used, simply as a buffer to wash cells with an isoosmotic solution, i guess it the pH won't really matter.

[biohazard]

Good point, MrMistery: as the natural environment of lymphocytes is blood, pH 7.4 is indeed their optimal pH in culture as well (at least according to this lymphocyte culture textbook I have). It also states that when phenol red is used as an indicator, pH 7.4 is salmon, 7.0 is orange and 6.5 is yellow.

This means that the cells can tolerate quite a bit more acidic conditions than their optimal. Alkaline conditions, though, seem unfavourable, because when the indicator is bluish (=alkaline), cell growth is virtually always bad.

Also, like MrMistery mentioned, if we're talking about PBS or such solution that is not used to actually grow the cells or not used to make growth medium, pH doesn't matter that much - the solution just needs to be physiological in terms of ionic concentration and the pH somewhere around neutral.

[MrMistery]

you also need to take into account the fact that the high CO2 concentration in the incubator and(more importantly) the CO2 produced by the cells in the process of cellular respiration will make the pH go down a little if it is too alkaline. I remember we used to use growth medium even if it was a little alkaline, for the before mentioned reason.

[biohazard]

Yeah, that's true: the incubator's CO2 pretty quickly restores an alkaline medium to better suitable pH. Room air usually turns medium bit alkaline if it's exposed for some time. This doesn't harm the cells if you just restore them to incubator and do not let them stand in your hood for the rest of the day

What I meant with alkaline medium and poor growth is that when your cells cannot "turn" the medium acidic by their respiration, it usually means they are growing very slowly, if they are growing at all (or they are in very low density)

Actually, I don't think I've ever discarded fresh medium just because it's gone alkaline, the incubator & growing cells' metabolsim fixes that in no time. However, cells in a well that has it's medium not turning gradually towards yellow/orange are usually too weak to be of any use in my experiments.

[DoctorStein]

Hey thanks for the reply, guys I mostly work with spermatozoa which prefer pH 7.4 to keep them swimming happily I really like the term "...my cells grow quite happily" ihihihii Now I need to work with thymocytes so I need to know their preferences. Well, then I think it is correct if I will use 7.2 or 7.4, right?

About this experiment, I am preparing a labwork for student to learn counting cells from this and that. Since thymus contains nearly homogenous cells, I want to use that organ. Yes, here PBS is used to wash the cells, and as a temporary medium while they are counting. The nest point of the experiment is to distinguish living cell from dead cell by supravital staining so I need them alive for some minutes before applying the dye (trypan blue). I think the culture medium is not necessary in this simple experiment. Or?

[biohazard]

I'm pretty certain that the cells stay ok in PBS for several hours, and longer if kept in cold as well. pH 7.2 - 7.4 should be just fine.

I have stored cells in cold PBS successfully for some 2-3 h at least, in PBS with some FCS or AB serum they should last much longer. For overnight storage in +4C I usually switch to culture medium, but I wouldn't be surprised if the cells were okay even in PBS+FCS or some other such solution.

[DoctorStein]

I think I will use PBS 7.4 instead of HBSS to soak my thymocytes, as the staining (trypan blue) is dissolved in PBS. Or is that ok to keep them in HBSS and then pour the dye, which is in PBS environment?

[MrMistery]

i don't think there's a difference, but substituting the HBSS for PBS would be ultra-safe