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Outline


  • plasmid-based protein expression in insect cells
  • plasmid containing the desired open reading frame(s) is directly transduced into insect cells via complexation with the polycation polyethylenimine (PEI)
  • plasmid gets diluted with every cell division, resulting in a likewise limited timeframe for protein expression
  • fast expression (approximately one week)
  • high viability of insect cells is maintained
  • high yields for intracellular and secreted proteins


To Do


please ensure the following is at hand and prepared in sufficient amounts

  • PEI40 (1mg/mL in ddH2O)
  • ExpiSf9 cells at 2 - 3x106 cells/mL
  • ExpiSf9-CD Medium (Gibco, 2068298)
  • Plasmid of interest, 1µg per 1x106 cells/mL


Plasmids Used




Flask Type125mL250mL500mL
Culture Volume25-35mL50-60mL100-110mL
Shake Speed95 +/- 5 RPM (50mm shaking diameter in Infors Minitron)
Used


table 01 - correlation between culture volume and flask volume


Day 1,


  • seed ExpiSf9 at 0.7x106 cells/mL in 25mL ExpsiSf-CD Medium in 125mL flask, from continuous culture
  • let grow for 1-2 days up to 2 - 3x106 cells/mL

Comments:


Day 2/3,


  • check cell density for 2 - 3x106 cells/mL
    • if the cells did not reach the density needed, let them grow another 24h
  • thaw your plasmid and PEI40 before starting the transfection
    • incubate the plasmid at 60°C for 10min prior transfecting, to minimize contaminations
    • DNA:PEI40 are added in a ratio of 1:4
    • the amount of DNA is always calculated to the total cell density
      • e.g.: 5x106 cells/mL in a volume of 7mL are 35x106 cells, therefore 35µg of DNA are needed
      • based on this you can calculate the amount of PEI needed
  • calculate the volume needed for 1/5 end volume of your flask and a density of 5x106 cells/mL
    • e.g.: 125mL flask, 35mL volume
    • calculate  with (5x106 cells/mL * (35mL / 5)) / your given cell density
    • e.g.: (5x106 cells/mL * 7mL) / 4.2x106 cells/mL = 8.33mL
    • transfer 8.33mL of your cells into a 50mL falcon tube
    • spin down and resuspend in 7mL for 5x106 cells/mL density


  • transfer calculated volume of cells into 50mL falcon tube - sterile
  • spin down 180xg for 4min
  • aspirate used medium and discard
  • resuspend in fresh ExpiSf-CD Medium to a density of 5x106 cells/mL in falcon tube and transfer to a flask
    • as 4x the volume of Medium is added in the end, please calculate the endvolume now and use a proper flask type (see table 01)
    • e.g. 6mL of 5x106 cells/mL will have another 24mL medium added in the end
  • mix the heated DNA with 500µL of ExpiSf-CD Medium, to cool the DNA down, but also do dilute it
  • add the DNA slowly but steadily to the resuspended ExpiSf9
    • swing or swirl the cells properly immediately
  • add PEI40 directl after adding the DNA
    • again swing or swirl the cells properly immediately
  • incubate the mixture for 3h at 27°C and 95 RPM (+/- 5 RPM, see table 01)


  • after 3h add 4x the volume of ExpiSf-CD Medium
    • make sure to use an adequat flask type for the amount of medium used

Comments:






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