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.
- Use of Real-Time Polymerase Chain Reaction to Measure Gene Expression in Single Cells
- Isolation, Culture, and Potentiality Assessment of Lung Alveolar Stem Cells
- 兼性异染色质(facultative heterochromatin)
- Isolation, Expansion, and Characterization of Human Islet-Derived Progenitor Cells
- 大鼠白细胞介素2(IL-2)酶联免疫分析
- The Zebrafish: An Overview of Its Early Development
- Methodology, Biology and Clinical Applications of Human Mesenchymal Stem Cells
- Assessment of Cardiac Conduction: Basic Principles of Optical Mapping
- 小泡运输(transport by vesicles)
- Derivation and Characterization of Hematopoietic Cells From Human Embryonic Stem Cells