AIM/HYPOTHESIS
With the new Multitron we are able to produce large-scale suspension cultures. The aim of this experiment was to steadily grow HEK293TLD cells in suspension and transfect them with both MT302 and LD401 plasmids for further proteomic and/or structural LINE1 studies.
A detailed logbook including cell counts and passages can be found here.
I also tried using the Luna for cell counting. It gives a pretty solid estimation of your cell numbers, so to use it for getting an approximate amount is does its job. However, I also found that when you have a small number of samples to count, the hemacytometer is just as fast, and probably more reliable.
MATERIALS
| Name | Company | Cat. nr. | Date received/made | Comments | ||||
|---|---|---|---|---|---|---|---|---|
| Culture | ||||||||
| Multitron | Infors | |||||||
| HEK293TLD cells | ? | Cells taken were stocked by Leila at 20M cells/vial. They were selected with blasticidin before stocking | ||||||
| Freestyle 293 expression medium | Gibco | 12338018 | Supplemented with; 1% FBS, 2mM L-glutamine, 2mL 0.5% Phenol Red solution, no p/s | |||||
| Tet-free FBS | Sigma-Aldrich | F2442 | ||||||
| L-glutamine | Gibco | 25030081 | 200mM (100x) stock | |||||
| 0.5% Phenol Red solution | Sigma-Aldrich | P0290-100ML | ||||||
| Polycarbonate Erlenmeyer flasks, 125mL | VWR | 89095-260 | This was used to sustain a maintenance culture | |||||
| Polycarbonate Erlenmeyer flasks, 1L | VWR | 89095-284 | ||||||
| Transfection | ||||||||
| Hybridoma-SFM | Gibco | 12045076 | ||||||
| PEI Max (MW 40,000) | Polysciences | 24765-1 | ||||||
| MT302 plasmid | Taylor et al., 2013; 3584 µg/µl | |||||||
| LD401 plasmid | (Taylor et al., 2013); 3009 µg/µl | |||||||
| Doxycycline | 5 mg/ml stock | |||||||
| 250mL centrifuge bottles |
METHODS
Cells that were thawed from stock were blasticidin selected just before freezing, so should contain the Tet operator. The cells were firstly cultured in a 125 mL flask for about a week. After that cells were passed to two 1L flasks, both containing 400mL medium, and grown until ~3 million cells/mL. Cells were then transfected according to the protocol, split 1:4 the next day and grown again up to ~5 million cells/mL. Cells were subsequently induced with 1 µg/mL doxycycline and harvested exactly 24h post induction. Yield of MT302 was ~15 grams due to mistake when making BBs, yield of LD401 was ~19 grams.
An a-ORF1 IP was performed to check the expression of LINE1 (Immunoprecipitation with antibody conjugated Dynabeads from cell powder). MT302 light and LD401 heavy powder were used as controls.
RESULTS
DISCUSSION
Expression levels are definetly lower compared to the control samples. There seems to be a faint band however, so there could be some expression, but it might very well be endogenous. I used the 'split-back' method as described in the book chapter this time, this may be detrimental to transfection efficiency.
Also, in the new cell powder there is a clear band at ~50 kDa, around the height of the IgG heavy chain. Although all samples were handled in parralel and equal, it could be that due to a lack of ORF1, the antibodies were eluted from the beads instead of the protein from the antibody. To test this, I will add a control with a-ORF1 beads only during elution.
The next thing I will do is move back to smaller scales to test the transfection method and test without the 'split-back'. Given the fact that I only used 1/20th of the required amount of DNA and PEI max due to the error in the protocol, I have sufficient confidence that it will work next time. Therefore, I will leave out the GFP control to check expression for now.