I found a couple of good protocols for this purpose: One from Dr. Montefiori's Duke Central Immunology Lab and one from Springer (attached).

The other one:

I used PBMCs from healthy uninfected donors.

Medium: RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

To stimulate PBMCs (uninfected): ad phytohemagglutinin (0.5 μl/ml) for 2 days.

After stimulation, replace phytohemagglutinin by 20 U/ml interleukin-2

Medium change: half of the volume every two days

Adding fresh PBMCs is dependent on time your Assay will run.

For up to 3 weeks no change should be necessary.

Otherwise just inoculate 1 Million fresh PBMCs with your (3 week old?) culture

The HIV culture protocol in my Phtesis is robust and sensitiv. You must remove the suppressor cells!

I believe that these papers can help to you (they are available for all):

Aquaro S, et al., Antimicrob Agents Chemother. 2000

Scopelliti F, et al., Antiviral Res. 2011

The clinical evolution in HIV infection results from immunosuppression characterized by T cell anergy, apoptosis ant chronic activation of T cells. The cell death and apoptosis are not directly linked to virus isolate when PBMC derived from patients were in vitro activated. II want to suggest you to use TFP molecule (TFP). This small and available molecule can restore the proliferation of T lymphocytes without antiviral effect [A. Achour et al, Virology, (2003) 315, 245-258]. In this paper you will find all the details of the cell culture conditions.

Yes, I saw those protocols and it's one of the reasons I'm asking here if someone has an expertise in it as there are so many different ways of doing it. And of course I am depleting CD8 from PBMC, whether they're from healthy donors or patients.

But thanks anyway.

It is easier to add a chemical product than depleting CD8 T cells. By using depleted CD4 T cells, even from healthy donors, you can expect an allogenic immune reaction characterized by a production of inflammatory cytokines.

Raphaelle

We use 30 IU/mL of IL2, 1x10^6 cells/mL to culture the cells. For activation, we use 1 bead/cell of anti-CD3/anti-CD28 Dynabeads. Once activated for three days, we change the media an the following days post activation: Days 3,4,5,7,10 then every three days keeping the above culture conditions. We do not add new cells to our culture. If you are doing a co-culture assay, then you will need to optimize. The cells lose their activation markers by about day 14 post-activation. I hope that helps.

Laura

I've used David Montefiori's protocol to grow primary isolates for the last 15 years. As a graduate student I traveled to his laboratory to learn neutralization assays. Been using it ever since with terrific results.

One thing I do often lengthen is the time of PHA stimulation. I often go 2 days or until the blasting T cells look like bunches of grapes. Then infect them with a high titer stock overnight and expect 60 ml of high-titer stock from 2.5x10^6 PBMCs. Best to use fresh cells.

Good luck.

I have worked on that area you can get my publication on Isolation and Biological Characterization of Non-B HIV Type 1 from Kenya

I prefer PBMCs without any depletion, an optimization was done to see T-cell blast formation i.e. PHA-P stimulated for 3 days at 3 ug/ml. I always used freshly isolated PBMCs. However if it is for virus production, I will always prefer HEK-293T transfection with appropriate plasmid.

All the best.

I have been using 100U/ml IL-2 for PBMC culture. For PHA stimulation, you can try use higher concentration for shorter time.

start with normal donor PBMCs and stimulate with(PHA 5ug/ml) for 48 to 72hrs. and to fresh donor PBMCs and add IL2 (100IU/ML), ideally both donor and patient PBMCs should be in equal volumes. I also prefer PBMCs with no depletion are just fine as well. Can do this depending with your volumes in T25 Flasks(preferably vented or with loosened caps for non vented)or 48 well TC plates. You can add fresh PBMCs every 3 to 4 days harvesting supernatant as well as adding fresh PHA prestimulated PBMCs for 21- 28 days (this can be synchronized to be once per week for 3 to 4 weeks). Medium to use is complete RPMI 1640 enriched with 10% FCS