PBMCs proliferation in hiv + patients - PBMCs proliferation (Jun/06/2005 )

hi

i m working on CMI in hiv positive patients, basically after stimulation with specific antigens.
i wanted to know that if there is any preservative for keeping pbmcs for proliferation to b done later, the max time duration for keeping, effect on the efficiency of pbmcs to proliferate and whether it will b feasible becoz in hiv + blood pbmcs r already low.
Actually i get the samples with much difficulty and i hav to preserve pbmcs becoz i dont hav the Ag right now.
can anybody help me?

Dear Priya,

Hi. We had lab experience with PBMCs for Dengue and HIV patients too. Sometimes nurses send the sample right after office hour. So, the sample was leave overnight in +4C. Even though, we still able to get a good cell count. I think is very important to know exactly how you isolate the PBMCs. Because we had found out different methods of isolation will give different effects. Please state if you free.

But, a few precaution steps we would like to share with you.

1. We use culture medium to do all the washing steps. Because our previous protocol which using the PBS wasn't too good. Might due to the pH. But, you need to monitor the pH of your cultute medium. Try to use a 100ml bottle to store it. because the air trap in the bottle may easily cause oxidation.

2.Once you had started the isolation proses, try to finish it within an hour or two.

3. Try to use Mr. Frosty to prevent shock freezing condition. Fill in isopropanol inside. Prior your isolation, try to keep Mr. Frosty in the -20C freezer. Once you had get the cells in the freezing media, store the vials in that Mr. Frosty and keep inside -80C freezer staight away for overnight. The next morning transfer the vial into liquid nitrogen tank.

4.Our freezing media is make up of fetal calf serum with 10% DMSO(without culture medium).

5.You may carried out a vaiblebility check after a week. Meaning to compare the cell count before storage and the cell count after a week.
You may also check the second or third vial after few weeks or months.

From the viablebility chech you may know is that neccessary to modified your protocol or not.

Another question that I wonder is :understanding of the antigen that you choosen. First of all, does the lymphocytesl only response to those antigen which are presented by MHC molecule. Do you need to express the antigen with a HLA or you need not to do so. Because from the theory of immunology we knew the CD4 cell will only get into efector phase(such as proliferation) when encounter with an antigen presented by MHC class II.
Please correct me if I am wrong.

Good luck
Hadrian.Trajunas