XC Assay of MoMLV Virus Stocks

Materials Wildtype MoMLV virus aliquot.  Stored at -80ºC. Medium:  DMEM + 10% FBS NIH 3T3 TK- cells XC cells Polybrene 1000x stock = 4 mg/mL, sterile filtered. U.V. light source on a stand. Procedure

Day 0:  Thaw XC cells and NIH 3T3 TK- cells. Day 2:  Plate NIH 3T3 TK- cells at 5x105 cells / 6 cm dish.  Prepare 2 dishes for each virus sample to be tested. Split and passage XC cells. Day 3: Refeed NIH 3T3 TK- cells with medium + 4 µg/mL polybrene.  In one dish add an equivalent of 1 µL of virus and in the second dish add an equivalent of 0.01 µL of virus diluted in medium.  Day 4: Split all the infected cells 1:20.  Day 6: Split XC cells for use tomorrow. Day 7: Suspend XC cells at 106 cells / 4 mL for each dish.  Aspirate media off of NIH 3T3 TK- dishes.  U.V. irradiate with lamp on stand over open dishes for 20 seconds.  Add XC cell suspension on each dish.

MF Note: According to Bass and Turner (see refs.) the ideal U.V. dose is approximately 0.018 J/cm 2 or a reading of 180 (µJ/cm2 x100) on a Stratalinker1800 which takes ~6 seconds due to an output of 3 mW/cm2 in this device. Day 9: Aspirate off media.  Stain cells with Coomassie blue. Maloney virus causes fusion of XC cells to form syncitium.  Count the number of these plaques on each plate with an inverted microscope.  PFU/mL = (plaques/plate) x (20 cell split)  / mL virus (10-3 or 10-5 ).  For example 500 plaques / plate from 1 µL virus = 107 PFU/mL. MF note: Cells should probably be fixed with formalin and washed with PBS before Coomassie staining.