I transfected px458 in Expi293F HEK cells with lipofectamine and then sorted single cell by FACS into 96 well plates.
The medium is Expression medium without antibiotics.
The cells are cultured in a 37C, 8% CO2 incubator with shaking.
However, after one month, no cell proliferation has been observed.
If anyone has done transfection with Expi293F and can tell me the solution or protocol, I would appreciate it.
When the cells are alone they have it very hard to grow, even without toxic/harsh procedure like FACS.
Does the plasmid have marker for selection?
Dear S Nakazawa!
Please you look at the articles:
Article A Transient CHO Expression Platform for Robust Antibody Prod...
(attached)Results Transient transfection in ExpiCHO-STM achieved high protein expression titers comparable to those in Expi293FTM -We transfected both ExpiCHO-STM and Expi293FTM cells with vectors coding (Fig 1).
High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines (attached)
2.5. Transient Transfection of Suspension Cells Transient transfection typically requires 10–15 days from culture expansion to purified protein (Figure 2). EXPI293F cultures with >95% viability were centrifuged and resuspended in fresh medium at 3 × 106 cells/mL within 1 h prior to transfection. In this case, cells were transfected using the ExpiFectamine 293 Transfection Kit according to manufacturer’s instructions. DNA was diluted in 5% of final culture volume while in a separate conical tube, ExpiFectamine was diluted in 5% of the final culture volume to achieve a final culture concentration of 1 µg DNA/mL and 2.7 µL/mL, respectively. Diluted mixtures were incubated for 5 min at room temperature (RT). DNA mixture was added to ExpiFectamine mixture and incubated at RT for 20 min before addition to cells. Twenty hours post-transfection, 5% and 0.5% of final culture volumes were added of Enhancer 2 and Enhancer 1, respectively, as per manufacturer’s instructions. As an example, 500 mL of final culture volume requires 500 µg of DNA diluted in 25 mL of medium and 1350 µL of ExpiFectamine diluted in 25 mL of medium. After 20 h, 25 mL of Enhancer 2 and 2.5 mL of Enhancer 1 are added to transfected flasks. Protein expression in culture supernatant was confirmed 48–72 h post-transfection and harvested 7 days post-transfection or when viability reached
I didn't work on HEK, but i tried to sort single K562 cell into 96 well plates, and recovered 84.5% positive clones.
To me, the main stress for these HEK cells come from transduction reagents. After transfection, wait a few days for the cells to calm down and start to grow, and then sort cells into 96 well plate may be helpful.
Dear Adam Flgarski
My plasmid is px458, which includes eGFP.
So I sorted GFP expressing cells with FACS.
Dear Alexandr Chernov
Thank you for this paper.
I read it, but this author didn't do single sorting with FACS for Expi293F HEK cells.
My vector includes Cas9 and gRNA to knock out specific gene.
So I need to pick up a single cell, but unlike normal adherent HEK cells, I cannot pick up a single colony in a floating cell.
Dear Shiqiu Xiong
Thank you for your suggestion.
I already tried your suggestion. After transfection, I cultured these cells for 7 days.
Because these cells still showed GFP expressing, I sorted with FACS.
Have you tried also on nonfluorescent but sorted cells?. The "GFP" group of protein is quite toxic for the cells, and phototoxic to.
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