Protocol transfection ExpiCHO cells, written by Rosa Schier

Day before the transfection:

• Count the cells: 60ul trypan blue (from sigma, dilute 2/3 trypan blue and 1/3 PBS 1x) + 20ul cells = 4x dilution to put on the Neubauer plate
• Calculate how much cells you have to centrifuge (6min at 1400rpm) to have a concentration of 2,5x106cell/ml, vacuum the supernatant and transfer to fresh ProCHO5 media supplemented with 4mM Glutamine

Day of the transfection:

• Heat up the DNA 15min at 65°C (once heated centrifuge 1min at max speed)
• Count the cells to know the concentration ex: counted 473 alive = 4,73x106cell/ml (473/4(4 fields of the plate) x4(for the dilution) x10’000(for the factor of the plate))
• For a transfection of 10ml (example) you need: 5x106cell/ml x 10ml = 50x106cells
• 50x106cells / 4,73x106cell/ml = 10,6ml of culture to centrifuge 3min at 1000rpm (little centrifuge) 6min at 1400rpm (big centrifuge)
• Resuspend the cells in 10ml of ProCHO5+Glut(4mM), put in the right erlenmeyer
• You need 3mg of DNA for 1L of transfection, put in the resuspended cells and mix (attention to put it in the cells and not on the wall of the erlenmeyer) ex: 30ug for 10ml of transfection
• You need 15ml of PEI-MAX (stock 1mg/ml) for 1L of transfection, put it in the resuspended cells and mix (attention to put it in the cells and not on the wall of the erlenmeyer) ex: 150ul for 10ml of transfection
• Put to shake 1h at 31°C (120rpm if it’s in an erlenmeyer, 180rpm if it’s in a maxi spin tube)
• After 1h add 2% of DMSO (dimethyl sulfoxide) and incubate at 31°C
• End of the transfection after 3 days if the protein is in the pellet and after 7 days if secreted into the supernatant

End of a transfection pellet:

• Take a sample to count the cells during the centrifugation alive/(alive + dead) x100 = viability%
• Put in maxi spin tubes and equilibrate
• Centrifuge 20min at 2400rpm
• Vacuum the supernatant (pay attention not to vacuum the cells)
• This is only for big pellets not for pellets from 10ml culture (go to the last step). Add 25ml of PBS (1x cold) to resuspend the cells and transfer into an 50ml Falcon tube
• Put the quantity of PBS needed to fill up the Falcon in the maxi spin tube to get all of the cells
• Centrifuge the Falcon 20min at 2400rpm
• Vacuum the PBS if the cells are well at the bottom (if not centrifuge again)
• Weight the pellet and freeze the cells in dry ice

End of a transfection supernatant:

• Take a sample to count the cells during the centrifugation alive/(alive + dead) x100 = viability%
• Put in maxi spin tubes and equilibrate
• Centrifuge 30min at 3200rpm
• Prepare a yellow glass bottle (check the bottleneck) + filter
• Pour the supernatant on the filter and put the pump on the filter
• Label the bottle and put in the fridge at 4°C