< D. Miller 12/96
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
1. Vaclave Klement, et al. PNAS 1969 63:753-758. Mixed culture cytopathogenecity: A new test for growth of Murine Leukemia Viruses in tissue culture.
2. Bass LR, Turner W. Appl Microbiol. 1972 Jan;23(1):200-1. Semi-micro XC cell assay technique for murine leukemia virus.
3. Johnson GS, Friedman RM, Pastan I. J Virol. 1971 Jun;7(6):753-8. Analysis of the fusion of XC cells induced by homogenates of murine leukemia virus-infected cells and by purified murine leukemia virus.
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/cm2 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.
ReferencesFor 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.
1. Vaclave Klement, et al. PNAS 1969 63:753-758. Mixed culture cytopathogenecity: A new test for growth of Murine Leukemia Viruses in tissue culture.
2. Bass LR, Turner W. Appl Microbiol. 1972 Jan;23(1):200-1. Semi-micro XC cell assay technique for murine leukemia virus.
3. Johnson GS, Friedman RM, Pastan I. J Virol. 1971 Jun;7(6):753-8. Analysis of the fusion of XC cells induced by homogenates of murine leukemia virus-infected cells and by purified murine leukemia virus.
