Hi

I presume you are setting up a one way MLR using irradiated stimulators and unirradiated responders.

To check the proliferation status of your irradiated cells you could set up wells of irradiated cells labelled with a tracker dye, stimulate with the mitogen conA and use flow cytometry to check the proliferation status, if the level of irradiation is correct there should be no no stimulation of the irradiated cells to proliferate. Once you have determined the radiation dose required to block proliferation you can then use unlabelled stimulators in the MLR assay.

To assess the proliferation of the unirradiated cells you could use a tracker dye to label the cells and follow the number of divisions using flow cytometry. This would additionally allow you to distinguish between the stimulators and responders as the stimulators would be unlabelled.

The adherent cells are probably macrophages /monocytes, which though important in the response should not affect outcome of assessment of proliferation as the T cells should not adhere.

As a rule I include as controls wells of responders alone to assess background proliferation and responders + ConA to assess maximal proliferation and to demonstrate that the cells are able to proliferate.

If you are finding large numbers of dead cells it is possible that the method of preparation of the spleen cell suspensions is not gentle enough to preserve viability. I find teasing is the best method. Also remember the large number of red cells present which need to be excluded from analysis. I do't know if you remove red cells prior to culture. I have found red cell lysis is detrimental and that density gradient separation is best and just living with any remaining red cells.

hope this helps

kay

Hi, you didn't mention whether you cleared RBC before you set up the experiment for proliferation assay. Debri from RBC interferes with proliferation studies. 

As explained by Kay, T cells and some small percent DCs and B cells also floating cells while Macrophage/Monocytes firmly adhere to the surface. Also, often fibroblasts will grow as you prepared cell suspension from spleen. If you are certain with the kind of cells that you are looking for (eg. T cells), I would suggest purifying them first. If you are interested in total splenocyte proliferation, you can use a non-radioactive but efficient method "CyQuant NF Cell Proliferation assay" where it will quantify total DNA content. Using this method, you can directly quantify the proliferation of all cell-types in a 96-well plate.

Hi Kay & Loka, thanks for the reply.

For checking proliferation status, I think it will help a lot. I'll try to search for the method.

For RBC, I lysed it with ACK buffer and the pellet looked fair and there were only a few of dead cells available when observed under the microscope. The dead cells appeared after 2-3 days of co-culture before I pulsed with BrdU. 

May be I can start with non-radioactive assay as mentioned by Loka to check the cells proliferation first to confirm which doses of irradiation and cell number are correct. 

Thanks again for the ideas.

Have you tried using plates that are not coated for tissue culture? I have routinely used bacterial plates for splenocyte cultures and untreated multiwell plates have become increasingly available over the last several years.  Usually the cells can be easily resuspended by pipetting without any enzymatic treatments altough using PBS without Ca, Mg also helps.

Hi Diana,

Thanks for the suggestion. I haven't tried to culture cells in non-coated plate. May be it will be a way to reduce the cell attachment on plate. 

Hi,

You cells shocked and according my experiences on this subject you should enrich your culture media (may be!!).

Thanks Mehdi . I have solved the issue. :)

Hi Tan Eng Chong,

You said above that you solved the problem.  What worked for you? We would like to know.  Thanks.

Hi Diana,

I just changed x-ray to mitomycin c. All stimulator spleen cells no longer attach or proliferate after treated with the inhibitor. So, now I can determine the responder spleen cells proliferation in mixed lymphocyte reaction. Hope I answer your question. 

hi Tan,

on a different note - could you please tell me how did you get "many splenocytes attached on the place after I re-suspended it before I proceeded to flow cytometry analysis"- e.g what kind of plate (coating?) and medium did you use? Briefly, my aim is to attach as many types of spleen cells as possible. Thanks in advance!

P.S I have seen that you have used radiation for attachment, but do you have some tips how to attach "healthy" and untreated splenocytes.

 Hi Dimova,

I think it is impossible to let the splenocytes to attach as many as possible because it is a mixture of different cell types. My previous experiment was to stop its (stimulator) proliferation so that the proliferation of responder cell can be determined. 

For your aim, I'm not pretty sure how to improve the number of attached splenocytes but according to my previous experiment, the number of attached cells increased after prolonged culture period. It must be the apc.

Hi

it seems you have two questions here:

1 how can you check that irradiated cells are no longer proliferating

2 how can you prevent cells adhering/dying between harvesting and flow.

1 to check that the irradiation dose is sufficient to prevent proliferation

use a cell tracker dye to label cells,

irradiate

then stimulate with a mitogen eg ConA or PHA for 3 to 5 days.

remember to use labelled unstimulated cells and unirradiated labelled cells as controls

harvest cells and assess proliferation by flow.

if irradiation dose is correct there should be no proliferation

2 A proportion of irradiated and unirradiated cells will adhere to the plastic during culture, however these should be APC which will not have divided during the MLR.

I think possibly the dead cells you are harvesting maybe the irradiated cells.

To set up the MLR

use unirradiated cells labelled with a cell tracker dye,

use unlabelled  stimulator cells or   label with a different cell tracker dye

this will allow you to distinguish the live responding cells from the irradiated stimulator cells

include unstimulated  and mitogen stimulated responder cells as controls, this will demonstrate the fluorescence of non-dividing cells and that the cells were cabable of dividing respectively.

Harvest the cells using PBS (without calcium or magnesium) supplemented with 0.1%azide and 0.1%BSA or 2%FCS. Keep the cells in this PBS in the fridge until ready to run into cytometer. This should prevent cells adhering and prevent them from dying well awaiting analysis.

Hope this helps and if you need more advice get in touch

cheers

kay

thank you very much!