Testing 0.5-0.75ul 2020 Transfection Reagent Volumes for Increased GFP in Dv-1 Cells

Using the TransIT®-2020 Transfection Reagent which had the best transfection at 1ul for the previous test, now I want to test less than 1ul transfection reagent volumes. I am not using the other reagent in this test. I will try 0.75ul 2020 reagent and 0.5ul 2020 reagent.

20221209 Plating S2 and DV-1 cells

  • I want to use S2 cells again as a control to show that the transfection works
  • I need to use the same density I have used previously: 1.5 * 10^5 cells per well in a 12 well plate
  • Need to count cells and then separate
  • Plate layout:
  • This means I will need 3 wells of S2 cells (4.5mL total volume) and 9 wells of Dv-1 cells (13.5mL total volume)
  • S2 cells
  • I cell scrapped one flask from 11/29 and centrifuged it at 800rpm 2 minutes, removed the supernatant, and resuspended the pellet in 6mL of 10% FBS Schneider’s medium
  • 20ul from the resuspention was added to a hemocytometer and counts from each section of the hemocytometer were taken:
section quadrant 1 quadrant 2 quadrant 3 quadrant 4 section average
1 273 265 259 259 264
2 278 252 276 267 268
  • Each section was averaged, and the average of the two sections was taken
    • Total S2 average: 266
  • The cells per mL is the average * 10^4
    • 266 * 10^4 = 2,660,000 cells per mL
  • Now I need to dilute this so that I add ~1.5 * 10^5 cells to each well in 1.5mL aliquoits
  • I used M1V1=M2V2 to determine how much volume would be needed to dilute the entire concentrated liquid, and then scaled down for the volume I actually needed
  • (2.66 * 10^6 cells/mL)*(5mL) = (1.5 * 10^5 cells)(x mL)
    • x = 89mL
  • I don’t need this much volume, so I divided the original volume (5mL) and dilution volume by 5
    • Added 16.8mL medium to a new tube
    • Used 1mL of the concentrated cell suspension to that tube
  • These were mixed and added to the A1, B1, and C1 wells of PLATE 7
  • The rest of the diluted S2 cells were seeded into new flasks
  • Dv-1
  • I cell scrapped 1 Dv-1 cell flask, centrifuged them for 3 minutes at 800rpm, and removed the liquid. Then the cell pellet was resuspended in 5mL of 10% FBS Schneider’s medium
  • 20ul from the resuspention was added to a hemocytometer and counts from each section of the hemocytometer were taken:
section quadrant 1 quadrant 2 quadrant 3 quadrant 4 section average
1 310 365 336 294 326
2 377 357 286 344 316
  • Each section was averaged, and the average of the two sections was taken
    • Total Dv-1 average: 321
  • The cells per mL is the average * 10^4
    • 321 * 10^4 = 3,210,000 cells per mL
  • I used M1V1=M2V2 to determine how much volume would be needed to dilute the entire concentrated liquid, and then scaled down for the volume I actually needed
  • (3.21 * 10^6 cells/mL)*(5mL) = (1.5 * 10^5 cells)(x mL)
    • x = 107mL
  • I don’t need this much volume, so I divided the original volume (5mL) and dilution volume by 4
    • Added 25.5mL medium to a new tube
    • Used 1.25mL of the concentrated cell suspension to that tube
  • These were mixed and added to wells A2, B2, C2, A3, B3, C3, A4, B4, and C4
  • The rest of the diluted Dv-1 cells were seeded into new flasks
  • The plate was left to grow overnight in the 23 degree incubator

20221208 Transfection

  • I am using the same concentration of the pAc5 plasmid as I used previously
  • I checked the densities of the cells before doing the transfections. There seems to be similar amounts of cells in each plate, maybe a few more Dv-1 cells even though I tried to make them the same
  • S2
  • Dv-1
  • 2020 reagent was thawed at room temp and vortexed and spun down, so was the plasmid
  • All work was done in the cell culture hood
  • All reaction mixes were made up beforehand:
  • Tube 1
    • 400ul Schneider’s medium
  • Tube 2
    • 300ul Schneider’s medium
    • 3ul concentrated pAc5 plasmid
    • 3ul 2020 transfection reagent
  • Tube 3
    • 100ul Schneider’s medium
    • 1ul concentrated pAc5 plasmid
    • 3ul 2020 transfection reagent
  • Tube 4
    • 200ul Schneider’s medium
    • 2ul concentrated pAc5 plasmid
    • 1.5ul 2020 transfection reagent
  • Tube 5
    • 200ul Schneider’s medium
    • 1ul concentrated pAc5 plasmid
    • 1ul 2020 transfection reagent
  • All reaction tubes were pipette mixed genetly
  • Let tubes incubate in the hood for 30 minutes
  • After the 30 minute incubation, all experimental reagents were added dropwise to the appropiate wells
  • 100ul tube 1 to wells A1, A2, A3, and A4
  • 102ul tube 2 to wells B1, C2, and C2
  • 104ul tube 3 to well C1
  • 101.75ul tube 4 to wells B3 and C3
  • 101.5ul tube 5 to wells B4 and C4
  • Plates were gently rocked back and forth and placed in the 23 degree incubator to be checked every 24 hours for a few days

The plate was imaged ~every 24 hours with a scope capable of visualizing GFP. Images can be found here