detachment of cells - (Oct/30/2006 )
im working with HEK293 & RAW264.7 macrophage cell line.From last few days,few cells are detaching from the surface & flowing in media.I change media & next day,few cells are again floating in media.But the cells which are attached are growing well.Does it mean that my cells are contaminated???
Please help.
As far as I can say for the 293 cells, they are only semi-adherent if I remember right. Are you sure that you are being "gentle" enough with them???
But I assume if they were contaminated you would at least see some stress sign within the cells who have attached and ar growing.
Dear Minnee,
I would not worry about the RAW 264.7 as they can grow as suspension cultures. They stick aggressively to TC plastic, so we grow them in suspension in Techne Stirrer bottles.....no need to trypsinise them, just grow in media, spin and use. The floaters you are seeing could therefore still be viable.
However your HEK293's are a different matter. The floaters will be definetely dead cells. This could be due to a number of variables, including :
CO2 Concentration
FCS/FBS batches
Confluence of Monolayer
Low level bacterial CONTAMINATION
Mycoplasma contamination
Temperature fluctuations of the Incubators.
" If in doubt, chuck them out".............revive from frozen master stock and start again.
293 in late passages tend to be less adeherant. Also at a higher cell density, more are likely to come off. To improve the adherence of the cells, first, pool the floating and adherent cells in a tube and centrifuge at 1000 rpm for 5 min. Then, replate the cells on collagen coated plates (Becton Dickinson Cat#354401: 60 mm; #354450: 100mm). Use these plates for the first few passages. When the cell line's adherence improves, begin passaging cells on regular plates.
I would not worry about the RAW 264.7 as they can grow as suspension cultures. They stick aggressively to TC plastic, so we grow them in suspension in Techne Stirrer bottles.....no need to trypsinise them, just grow in media, spin and use. The floaters you are seeing could therefore still be viable.
However your HEK293's are a different matter. The floaters will be definetely dead cells. This could be due to a number of variables, including :
CO2 Concentration
FCS/FBS batches
Confluence of Monolayer
Low level bacterial CONTAMINATION
Mycoplasma contamination
Temperature fluctuations of the Incubators.
" If in doubt, chuck them out".............revive from frozen master stock and start again.
But according to the data sheet which we got with RAW264.7 cell line,these cells are adherent.So can they grow in suspension culture?
Dear Minnie,
We have grown both RAW 264.7 and J774 (both murine macrophages) in suspension culture for about the last 20 years. We have many papers in the literature, just look for Salvador Moncada or Adrian Hobbs...... Nitric Oxide field of research.
As stated already the advantages are :-
No need for purchasing TC flasks, the Borosilicate Stirrer bottles are reusuable...... some of mine are 20 years old.
No need to purchase Trypsin or PBS because you do not need to trypsinise them. Also saves on media costs.
Viability is greater as no trypsinisation.
Saves time in the lab....i.e. no requirement for trypsin.
Can grow vast amounts of cells for experiments in a small space.....no need for hundreds of flasks.