Testing Cell Transfection Reagents and pAc5 Plasmid on Primary innubila Cells

  • I am using a 12 well plate of innubila primaries that I made on 9/28. There are wells with “supernatant” in them, these are wells with a lot of debris from the primary generation process and they also have some cells. I did not use these wells but they are on the plate
  • Because I am limited on wells of cells, I am only going to use wells of just primary cells as the control. I will not get any wells that have transfection reagent only or plasmid only. Based off of the S2 transfection and cell counts from that I think this is fine
  • Plate layout:
  • For this I made only 2 master mix tubes
  • Plasmid DNA is at ~1ug/ul
  • All of this was done at room temperature in the cell culture hood. Transfection reagents were thawed at room temp, vortexed, and spun down before use
  • Tube 1
    • 200ul Schneider’s medium
    • 2ul concentrated pAc5 plasmid
    • 4ul insect reagent
  • Tube 2
    • 200ul Schneider’s medium
    • 2ul concentrated pAc5 plasmid
    • 6ul 2020 reagent
  • Each tube was pipette mixed gently
  • Tubes incubated in the hood for 30 minutes
  • Then the reagents were dripped into the wells, mostly in the middle because most of the primary cells are clustered into the middle of the dishes:
    • Added 103ul of tube 1 to wells B1 and B2
    • Added 104ul of tube 2 to wells C1 and C2
  • Incubated plates at 23 degrees for 24 hours then checked. The plate was always kept in the incubator between checks

20221020 Checking transfections

  • No experimental wells showed any evidence of GFP after 24 or more hours
  • I realized I should have used S2 cells as a positive control
  • There was a lot of autofluorescence in the primary cells, and it was about the same amount for control and treatment. I think this is due to the amount of debris/clumps of cells with primary
  • Some images from the plate are represented here