चूहा Splenocytes से टी सेल ग्रोथ फैक्टर की तैयारी

Thank you for a very interesting and useful protocol. I would be interesting in knowing if the cells that are pelleted after 48hours and discarded - are these considered T cells at this time and can these be subsequently expanded ?

Most of these cells are indeed activated T cells but this is not a pure population, I would recommend checking the % of CD3+ cells to know exactly what you have in your mix. If you want a clean T cell population you may need to do a separation by either magnetic beads or flow cytometry.

These T cells can be expanded after the 48-hr culture. I would recommend resuspending the pellet in fresh medium (same as described above) with no ConA but with 10% TCGF and keeping them in culture for 4-7 days before re-stimulating them with ConA (or any stimulus you want to use). T cells will divide a lot in the presence of TCGF so you will want to watch them and split them as they grow. You will probably need to split them every 2 days. The re-stimulation will work best if you give the T cells some irradiated antigen-presenting cells but you should get some stimulation with ConA alone and no antigen-presenting cells. During the first stimulation described above, non-T cell splenocytes will act as antigen-presenting cells but will be dead and useless at the time of re-stimulation. Each round of stimulation will increase your T cell %.

I hope this helps and good luck with your experiments!

Thanks for your response. Thats very helpful

Thanks for the previous response - I wanted to know a couple more things if possible - after 10 - 15 ml aliquots are made and placed in -20 :

(1) how much do you tend to use at a time ?

(2) are there any porblems with the sample thawing and then being refrozen?

We use the TCGF at 10% when growing T cells in the expansion phase so we use anything from 5 ml to 50 ml of TCGF on a given day.

I have never re-frozen thawed TCGF and would not recommend it but you can keep the thawed tube in the fridge for 10 days, this works well.

HI Christine,

Thanks for your help in answering some of the quesitons I have had. I have some more for you ! I have used the TCGF to expand a separate population of T cells. What I wanted to ask was

(a) when you are splitting the cells - what is your cell count approx -eg do you split if the cell count is greater than 1 x10^6 /ml

(b) in terms of the irradiated APC - what did you use and how did you go about doing this?

(c) if I was to use ConA alone to stimulate the cell line - would I need to use the methyl mannoside again to deactivate it after 48hours ?

Thanks for any  help you may be able to give

Rohit 

Hi Rohit,

Sorry for this long delay in ansering your questions.

(a) I seed the cells at 0.2 million/ml and usually need to split them after a couple of days, when they reach 0.6 - 0.8 million/ml. I try to never let them go over 1 million/ml.

(b) for rat T cell activation I use irradiated rat thymocytes (see the first steps of this video:

Beeton C., Chandy K.G. (2007) Induction and monitoring of adoptive delayed type hypersensitivity in rats, J. Vis.  Exp. 8, http://www.jove.com/index/Details.stp?ID=325)

For human T cells I use irradiated mononuclear cells (or purified B cells) from the same donor.

(c) No, you won't need to use the MM because in that case you are interested in the cells and not the supernatant, so you would throw the excess ConA away with the supernatant.

Christine

Hi Christine

Thanks for the reply! thats really helpful.

regards

rohit

Dear Christine,

I have got a question regarding the red blood cell lysis buffer you have used. I found in other protocols that they used NH4Cl in addition EDTA and KHCO3. Could you please let me know why you have just used NH4Cl. I am doing Lewis rat spleen culture for the first time and I am concerned about this red blood cell lysis step. Do you think there is a possibility that the red blood cells will cluster if not treated with EDTA.

Another question is regarding the cell strainer you have used (70u). I was planning to use a 40u cell strainer to avoid having cell clusters. From your experiance, please let me know about using a 40n cell strainer.

Regards,

Karima

Since we are interested in the supernatant and not the cells we have never really been worried about cells clumping. We haven't observed any significant clumping during this procedure but you can add EDTA to your solution, it will not drastically affect its osmolarity.

All the splenocytes are less than 15 um in diameter so a 40 um cell strainer will give you good results.

Regards,

Christine

What are the difference between Heat activated and non heat inactivated Fetal calf serum (FCS)? Please explain with examples.

Heat inactivation (56C for 30 min) of serum is done to degrade the complement that could induce cell lyzis in the presence of antibodies. This procedure was established several decades ago.

Heat inactivation may not be necessary, depending on the cell grown and the source/technique of serum production. I have never tried using non heat-inactivated serum as I don't want to change what is not broken and this has always worked well for us. If you are starting a new protocol I would certainly encourage you to test a sample of the same serum, before and after heat inactivation to decide on which works best for you.

Heat inactivation (56C for 30 min) of serum is done to degrade the complement that could induce cell lyzis in the presence of antibodies. This procedure was established several decades ago.

Heat inactivation may not be necessary, depending on the cell grown and the source/technique of serum production. I have never tried using non heat-inactivated serum as I don't want to change what is not broken and this has always worked well for us. If you are starting a new protocol I would certainly encourage you to test a sample of the same serum, before and after heat inactivation to decide on which works best for you.

Rat T-stim that we normally order from BD doesn't recommend freezing, but rather storing at 4'C. I would think freezing it would possibly increase the half-life whereas keeping at 4'C it would go bad within a matter of weeks. Do you think there's a big issue freezing and thawing, and why does BD not recommend it? Thanks

I don’t know why BD would not recommend storing TCGF at -20C. I have never used TCGF other that “home-made” as we always euthanized healthy rats with no other use for their spleen.

We have been very successful in storing this TCGF at -20C for several months but found that it lost efficacy if kept for more than 10-15 days at 4C. We try to aliquot the new TCGF before freezing but have also frozen it bulk (500 ml at a time) and then thawed, aliquoted and re-froze without loosing activity. I have never tried several cycles of freeze-thaw though and we try to prepare aliquots that can be used up within days of thawing.