10 ug/ml Dextran improves infectivity. Check out the David Montefiori SOPs

Yes, this improves infectivity in Macaque salivary tissues which are very difficult to infect.

Hi you can use polybrene to increase the infectivity and in turn more p24. I want to know the final aim of your experiment then i can suggest something better.

Thanks for all your suggestions. The aim for my experiment is to isolate RNAs from the infected primary CD4+ cells to perform Northern Blot, thus severe cell death will lead to poor quality of the RNAs. Now I am planing to perform a multiple infections, with infecting a fraction of cells first and followed by mixing fresh cells with the infected cells. Dose anyone has used this method and whether it is better than a single infection procedure?

I believe the others mean dextran sulfate, not dextran.

Cell death is often a function of the virus isolate you use. You might compare different isolates.

Do you have to use primary cultures? Consider the use of cell lines, some of which rapidly develop high levels of infection, with many more cells infected than in primary cultures. But with most cell lines, cell death does occur.

Dose anyone has used this method and whether it is better than a single infection procedure?

Yes. I had also used this method.

For me the better method is represented by cultivating PBMC infected by a single type of HIV (example HTLV-III-B) and isolated from one human patient without co-infections. How can You do?

FIRST STEP

Peripheral blood samples were collected from a patient-donor with HIV infection into 10 non‐heparinized hematocrit tubes. Minimal part of collected blood was processed for serum into tubes spun for 12 min at 12 000 RPMs in a 4 °C centrifuge, and then transferred to 0·5ml microcentrifuge tubes for storage at −80 °C until sent for analysis. Part of collected blood was destined to PBMCs isolation. PBMCs were obtained after Ficoll-Hypaque fractionation. After isolation and three washing with PBS (pH7.2, at room temperature), PBMCs were placed in culture with specific medium in in cell culture flasks. The minimum concentration of cells required was 250,000 cells / mL. The size of the cell culture flasks was chosen according to the number of cells obtained (Nunc, Lab‐Tek, Kamstrup, Denmark). Specific medium was composed by RPMI with glutamine and 10% serum of the donor-patient and 1,200 units/ml of IL-2. Cells were cultured in a Heraeus thermostatically controlled incubator at a temperature of 37 °C in an atmosphere containing a constant flow of 5% CO2 (v/v in air). 1/3 of culture medium was changed every 3 days. At day 12 (before of changing the usual part of culture medium) , 1 mL of supernatants was collected and tested by quantitative and qualitative PCR for ascertaining the infection.

At day 12, the HIV infection of PBMC culture was positive with high expression of virsus copies/ul. Then at day 15 without change of medium, whole supernatant of culture was collected and in part frozen at -80.

SECOND STEP

Peripheral blood samples were collected from a patient-donor without infection. PBMCs were obtained after Ficoll-Hypaque fractionation. After isolation and three washing with PBS (pH7.2, at room temperature), PBMCs were incubated with infected supernatant (collected as previously described) for 4 hours in a Heraeus thermostatically controlled incubator at a temperature of 37 °C in an atmosphere containing a constant flow of 8% CO2 (v/v in air). After incubation, cells were washed in free RPMI and then placed with RPMI-glutamine and 10% serum of their uninfected donor and 1,200 units/ml of IL-2 in an atmosphere containing a constant flow of 5% CO2 (v/v in air). 1/3 of culture medium was changed every 3 days. After 12 days, infection was tested by PCR. After ascertained infection, cells were continously-cultured in a Heraeus thermostatically controlled incubator at a temperature of 37 °C in an atmosphere containing a constant flow of 5% CO2 (v/v in air).

Limit your viral input at the moment of infection. Cells will remain in better condition and produce more infectious virus. From a practical point of view just try different amounts viral input.

Have a look at figure 2A in our paper : Pannecouque C, Daelemans D, De Clercq E.

Nat Protoc. 2008;3(3):427-34.

Best regards,

C. Pannecouque

Hello,

I want to suggest you to use TFP molecule (TFP). We shown that this small molecule can restore the proliferation of T lymphocytes without antiviral effect (from micromolar to nanomolar) derived from AIDS patients [A. Achour et al, Virology, (2003) 315, 245-258]. I recommend you to continually use the drug in the complete medium.

Best regards,

Ammar Achour

You may try spininfection (2900rpm, 2h, 37C) in presence of either Dextran sulfate or polybrene.

I would add fresh, uninfected cells to the culture every 2-3 days. It works for us when we want to produce high amounts of clinical isolates in CD4+ T cells. Also consider using viral isolates that are less cytopathic.

Three points you have to consider. 1. viral input, 2. availability of uninfected cells, 3. virulence of the viral isolates. lalitha kabilan

Hi,

I'm actually presently setting up a protocol to achieve this out of PBMC from HIV patients on ART treatment. I'm not sure it's the right place to ask this but 2 things seem strange to me:

- Personally, I activate the HIV-PBMC to induce viral replication but in all the protocols I find about virus isolation, people seem only to activate the PBMC from healthy donors they feed the culture with. Is there is a reason for that?

- My second regard (among so many!) is about IL-2 concentration: Luisa Genero, you advice 1200 IU/mL of IL-2 where I read 20, 50 or max 75 IU/mL elsewhere. Could you explain me the reason for such a high concentration?

Thanks!!