Hi everyone,

Firstly, thank you all for reading my concerns...

For my thesis project, I'm trying to standardize a co-culture with NK and autologous CD4+ T cells HIV-1 infected. For that purpose, I made negative selections with MACS columns (Miltenyi kit) for CD4+ and NK cells (viability and purity were around 94% for each population). After that, I followed a spinoculation protocol for the infection of CD4+ (1 million CD4+T cells plus 3ng of the virus, centrifugation= 2500RPM per 90min after that, I did two washes with PBS) At the beginning, I was struggling with the cell viability of CD4+ cells after infection.... I've tried so many things but so far, cell viability is around 70%. Nevertheless, I continued with the co-culture protocol thinking that PHA and IL-2 (8ug/mL - 50UI/mL) stimulation necessary for the infection was a little strong for these cells.

But now, I have another problem, I made three viral points evaluations by ELISA for the detection of p24 viral protein in the supernatants of CD4+ cultures and it was clear that the highest detection point was at 7 days after infection so, that's the point where I wanted to evaluate the NK activity against CD4+ HIV-1 infected cells; however, when I measured the p24 protein by ELISA (highest point of detection 200pg/mL) they are way too far of the highest point of the detection limit even after making dilutions of the supernatants(1:10; 1:50; 1500; 1:1000) so, my question is... it makes sense that the viral stock that was used for these experiments which concentration is approximately140ng/mL could be detected by the same kit of ELISA at 1:500 dilution but a higher dilution will be required for these experiments?

Am I missing crucial details for my purpose?

Thank you for your help, any advice will be very important to me!! by the way, sorry for my English, hope I've explained clearly my concerns.