Necessary information-
1. Target cell line: mutuDC/ DC1940 (mouse CD8a+ immortalized cells). Passage is over 25 (early passage not available). Cells are cultured in IMDM media (10% FBS, PenStrep, Sodium bicarbonate, HEPES and 2-mercaptoethanol)
NOTE: Highly sensitive cell line. Needed to be handled very carefully. siRNA mediated transduction is not feasible.
2. Plasmid: pLKO.1 plasmid with shRNA against a target long non-coding RNA (not commercially available so had to clone it myself).
3. Virus particles generated using CalPhos kit in HEK293T cells (cultured in DMEM). Virus collected at 24hrs, centrifuged and filtered using 0.45 micron syringe filter, aliquoted and stored at -80C.
4. Transduction: Cells are seeded at 0.15x106/ well in 12-well plates. After 12-14hr, each viral supernatant were added as follows: 50ul > 100ul > 150ul > 200ul. Total media in per well was 300ul. After 10hr of incubation, viral media was changed and fresh media was added. Media was changed after every 24hr as there was a lot of cell death (no extra supplement of FBS or other reagents were used in the media). After 72hr of transduction, Puromycin was added at the conc. of 0.5ug/ml (based on kinetics done earlier) in the same well where the mutuDCs were transduced (cells were not transferred to fresh plate as they will go under stress).
PROBLEM: After 4-5days of Puromycin selection, the (supposedly) transduced cells are proliferating well in 12-well plate. After each well was about 60%-70% confluent, the cells were dissociated (using non-enzymatic dissociation media; contains PBS and EDTA) and transferred into 6-well plate (from one well of 12-well plate to one well of 6-well plate). Within 14hr-16hr the cells started going into stress (swollen, serrated cell membranes, etc.) and after 24-30hr they started dying. I even tried transferring cells from one well of 12-well plate to two wells of 12-well plate (thinking maybe surface area could be the issue), but still faced the same problem. Now the cells are not under Puromycin selection as it might further add to the stress.
Kindly provide any and all suggestions as per your knowledge to help me out of this issue. Thank you in advance.
First of all, perform the infection experiment using freshly prepared virus containing medium without freezing the virus.
Freezing lentivirus at -80°C will reduce titers by a x10 or more.
Second, why is the virus-containing media collected and centrifuged before filtering? Because we are only removing cell debris by filtering, pass the virus-containing medium through the filter without centrifuging.
In gene overexpression experiments using lentivirus, freshly collected virus-containing medium will be diluted 1/4~1/8 for infection, but in KD experiments using pLKO.1, we use virus-containing medium with 1/2 dilution or undiluted for infection. For this protocol, HEK293T must me cultured in the same culture medium as target cells.
If you want to freeze lentiviruses, you can mix PEG-8000 and precipitate the lentiviruses with PEG as a cryoprotectant, preventing a significant decrease in titer.
Virus Precipitation Solution (PEG-it) is commercially available, but you can also make your own as described in the following paper.
https://www.mdanderson.org/documents/core-facilities/Functional%20Genomics%20Core/Homemade%204fold%20lentivirus%20concentrator.pdf
- Badges
- Science topic