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titration of lentivirus 4 H' g6 Y& I3 e) C4 u3 { 9 u9 I+ e3 n; X- X' w1. The day before titration, seed 0.5 × 10^5 293FT cells in 2 ml of DMEM-10 into all wells of a 6-well culture plate, ensuring a uniform spread of cells on the bottom of the wells. Prepare one plate for each vector stock to be titrated. Incubate overnight at 37°C, 10% CO2. 0.5 × 105 cells are seeded to produce 1 × 10^5 cells per well the next day. The number of cells seeded must be accurate because it will be taken into account when calculating the titer. 9 _# Z! L% F7 n" p/ G8 B0 e# W3 y % t W& F; c3 e* \% a3 H* |2. To five wells, add aliquots of the vector to be titrated: use 50 μl and 25 μl of the undiluted stock, and 100 μl, 50 μl and 25 μl of a 1:50 diluted stock (corresponding to 2.0, 1.0, and 0.5 μl of undiluted vector). Do not infect the cells in the last well; these are controls. Incubate 2 days. 0 v5 h# z `) F( I) _/ f+ y0 M; I7 B( v5 c4 G( }
3. A 1:50 dilution is obtained by diluting 5 μl vector into 245 μl DMEM-10. Since the added volumes are small relative to the culture medium (2 ml), total volumes are not corrected. / P9 ^# m4 F1 I2 p) Y! l' \; E4 |6 ^/ `/ O
4. Before the fluorescence-activated cell sorter (FACS) analysis, remove the culture medium, wash once with 2 ml PBS, and add 500 μl of 0.25% colorless trypsin/0.53 mM EDTA. Incubate 5 min at 37°C. Pipet up and down with a 1000-μl pipet tip to disrupt clumps. Transfer cells to a FACS tube containing 500 μl PBS. Cells will detach after a 5 min treatment with trypsin/EDTA. 1 s$ A8 Q* P. A/ P6 p7 t6 y7 [% N, L8 \
5. If desired, an aliquot of the cells can be kept in culture. If the FACS analysis is not done within 1 hr, cells can be fixed in 4% (w/v) paraformaldehyde solution for 30 min and kept for at least 1 week at 4°C.) @( k' B/ @) l
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6. Determine the percentage of GFP-positive cells by FACS analysis. " k/ }( `" _7 o, Y, g9 q7 ?6 c8 j/ E+ R! r6 g3 @5 r; j
7. Calculate the titer in transducing units (TU)/ml, according to the formula:Vector titer = 1 × 10^5 293FT cells × % of EGFP × dilution factor. For accurate titer calculations, the number of GFP-positive cells in 2 wells infected with 2 consecutive dilutions must be close to the expected 1:2 ratio. This linearity is observed when <15% of the target cells are transduced. 作者: naturalkillerce 时间: 2011-9-25 10:53