jacksonbear

Has anyone ever used Amicon Ultra centrifugal filters to concentrate HIV?  I need a way to concentrate my virus to improve the infectious titre of a molecular clone, and out BSL2+ lab does not have an ultrafuge in it.  Are there any alternatives to spinning the virus down that are effective at concentrating the titre?

I have been titering a batch of HIV on TZMbl cells using B-Gal staining.  I have calculated the TCID50 to be 5.6x10^4 for 100ul of virus, and calculated the FFU/ml to be 1.2x10^6.  It is my understanding that the concentration of infectious particles (pfu/ml or ffu/ml) should be approximately 0.7(TCID50).  Why am I getting a FFU/ml that is a log higher when TCID50 is adjusted for 1ml?

if it helps, this is what I did.

seeded TZMbl cells in quadruplicate in 96 well plate for 7 serial log dilutions starting with neat virus through 10^-6.  The following day I diluted my virus by serial log dilution, and added 100ul of each viral dilution to TZMbl cells in quadruplicate.  I infected the cells for 3hours, then washed off the virus, and incubated for 3 days.  After 3 days I washed the cells, fixed them and stained with X-Gal for 2 hours.  I then proceeded to count the number of blue foci in each well.

10^0  = all wells positive (too many to count)
10^-1 = all wells positive (too many to count)
10^-2 = all wells positive (too many to count)
10^-3 = 75, 86, 99, 122 blue cells/ well
10^-4 = 20, 10, 7, 20 blue cells/well
10^-5 = 0, 0, 0, 5 blue cells/well
10^-6 = 0, 0, 0, 0 blue cells/well

Thanks for any help/advice you can share.

Andrew

I am trying to quantify the TCID50 of a batch of HIV BaL that I have.  I infected TZMbl cells with 10 fold serial dilutions of my virus starting with neat virus for 3 hours.  After three days of culture I stained the cells using X-Gal and looked for blue cells.  My neat wells containing undiluted BaL  viral supernatants from T cells were all dead, presumably from the culture medium and not virus, as the next dilution had ~40% blue TZMbl cells.  Any suggestions on how I can calculate the TCID50 when I don't have any wells with 100% infected TZMbl cells?  I also ran the same experiment in parallel using IIIB viral supernatant collected from transfected 293 cells, which did not show any cell death with neat virus, and also did not have any wells with 100% infection.