[Large-scale real-time titration of green-fluorescence-protein-marked recombinant retrovirus: comparison with standard titration method].
OBJECTIVE
To compare large-scale real-time titration method (LaSRT) with standard titration method by flow cytometry (FACS) for determining the titers of green-fluorescence-protein (GFP)-marked recombinant retrovirus.


METHODS
(1) Standard titration method: NIH3T3 cells were inoculated at 2x10(5) /well in 6-well plate, and after cell culture for 12 h, 0.5 ml, 50 microl and 5 microl GFP-marked recombinant retrovirus (n=3) were respectively used to infect the cells, with the final concentration of polybrene being 8 microg/ml. Forty-eight hours later, the cells were treated with trypsin and assayed for the positive rate of GFP by means of FACS. When the positive rate was lower than 10%, the titer was calculated according to the equation: virus titer (TU/ml) =2x10(5)xGFP positive rate/volume of virus stock solution used. (2) LaSRT method: The cells were inoculated at 5,000 cells/well in a 96-well plate, and after cell culture for 12 h, 90 microl/well complete culture medium was used with 8 microg/ml polybrene and 10% newborn bovine serum, 10 microl virus was added into the first well, and ten-fold dilution of the previous virus-containing solution was performed before the virus was added into the next well (8 wells in each group, altogether 3 groups). Forty-eight hours later, inverted fluorescence microscope was used to observe the fluorescence-positive cells in each well. The virus titer was calculated according to the equation: virus titer (TU/ml) =mx10(n+1), where n is the serial number of the reference well, and m the number of positive cells. (3) LaSRT was used to study the influence of freezing/thawing on the titers of recombinant retroviruses.


RESULTS
The virus titer obtained with standard method by FACS was (1.54+/-0.38)x10(6) TU/ml, and that of LaSRT was (1.33+/-0.57)x10(6) TU/ml (P>0.05). After one cycle of freezing/thawing, the virus titer dropped to (18.1+/-9.9)% (n=7).


CONCLUSIONS
LaSRT is more rapid and convenient as well as easier to determine the virus titer compared with standard method, and no significant difference is found between the two titration methods.
Jiang
Qian-li
QL
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Wang
Jian-min
JM
Jiang
Shan
S
Wen
Li-min
LM
Zhou
Hong
H
D000818:Animals; D005434:Flow Cytometry / Q000379:methods*; D005822:Genetic Vectors / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis*; D051379:Mice; D041681:NIH 3T3 Cells; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins