小中大
滴度测定：Determination of virus titer by FACS analysis
1. Plate 1 x 105 NIH-3T3 cells per well of a 6 well plate 1 day prior to the assay.
2. Thaw a frozen aliquot of virus.
3. Prepare serial dilutions of viral supernatant (total volume: 500 l):
Neat: 500 l supernatant + 0 l medium
1/3: 166 l supernatant + 334 l medium
1/10: 50 l supernatant + 450 l medium
1/33: 16 l supernatant + 486 l medium
4. Remove medium from the cells and replace by dilutions of viral supernatant.
5. Add polybrene to each well at a final concentration of 4 ug/ml.
6. Add 2-3 ml pre-warmed medium to each well 4 h post-infection.
7. Incubate for 2 days in 5% CO2 at 37oC for maximal GFP expression in infected NIH-3T3 cells.
8. Trypsinize the cells and wash once with PBS 2% (v/v) FCS, and determine GFP-positive percentage by FACS.
9. Calculate titer as follow:
% GFP+ cells x number of cells/well (on day 0) x 2 x 1/dilution = virus particle /ml.
