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titration of lentivirus ; H3 z( i- o/ w+ j3 A. r * v9 A9 {" z5 t1. 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. : v+ N, i: e- n9 o, r l$ G: b6 g! | / N$ N7 R7 D( v9 q5 W/ ^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.2 ^2 s* Y3 d' Y
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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. / k5 |# X V7 | # B4 @8 f( b. C8 x3 H5 y% l$ y4. 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. 7 C' w1 n8 c! e) A$ k 0 Y# b: b. z( T% g$ |4 n2 u; |0 ]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.$ U1 N8 u! m0 V( K
6 H* z& ^* Z1 X* d! x* Y- f 6. Determine the percentage of GFP-positive cells by FACS analysis.; }4 _' _; R( Z: O: |$ r, e
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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