Akipp4: Created page with "'''Transduction Reaction''' * The volume of the reaction is based on the amount of virus available, the number of cells and the MOI being used * Prepare the mixture as follows: X uL of concentrated virus X uL of SF Macrophage Media (chose this volume based on the vessel the cells will be grown in ** 1000X polybrene '''Protocol''' # Count cells, add cells to appropriate size tube, spin down at 400 x G for 5 minutes # Re-suspend cells in the transduct..."

2022-01-11T17:16:43Z

Created page with "'''Transduction Reaction''' * The volume of the reaction is based on the amount of virus available, the number of cells and the MOI being used * Prepare the mixture as follows: ** X uL of concentrated virus ** X uL of SF Macrophage Media (chose this volume based on the vessel the cells will be grown in ** 1000X polybrene '''Protocol''' # Count cells, add cells to appropriate size tube, spin down at 400 x G for 5 minutes # Re-suspend cells in the transduct..."

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'''Transduction Reaction'''

* The volume of the reaction is based on the amount of virus available, the number of cells and the MOI being used

* Prepare the mixture as follows:
** X uL of concentrated virus
** X uL of SF Macrophage Media (chose this volume based on the vessel the cells will be grown in
** 1000X polybrene

'''Protocol'''

# Count cells, add cells to appropriate size tube, spin down at 400 x G for 5 minutes
# Re-suspend cells in the transduction reaction mixture
# Spin at 400 X G, 10 degrees for 1 hour 30 minutes
# Re-suspend the cells in the virus containing media and transfer to an appropriate size flask or plate, incubate overnight at 37 degrees overnight
# Transfer the contents of the flask to a new tube, spin down and replace the media with 500 uL of fresh complete RPMI
# Incubate plate for 48-72 hours at 37 °C

'''Notes'''

* Number of cells: Select number of cells that allows for an MOI of 300, some literature suggests MOI up to 500
* If making a cell line add selection media at the incubation period in step 6. I would also suggest comparing live/dead cell counts for both the control and the transduced cells before every media change to determine if the selection process is working

Transduction of THP-1s - Revision history