RAW264.7 Cells .. anyone familiar with this cell line? - need help please (Nov/16/2005 )
Hi, im new here. I desperately need help with culturing RAW 264.7 (a mouse macrophage cell line). Please bear with my long post..
I obtained this cell line from ATCC in July 2005 and then i was relatively new to cell culture so I didnt think too much except to passage, expand and freeze sufficient vials of cells. Left them aside until recently I have to thaw them because im starting on some transfection experiments.
The problem im facing now is poor cell morphology and viability They are adherent as they are supposed to be, but a mixture of elongated spindle shaped cells and circular cells that are usually granular looking. Some are large dome-shaped and dendritic looking even. They reach confluence very quickly although im very conscientious about splitting them at 80% confluence. They still look like that after several passages . I've tried maintaining them up to about P11 but they just look worse. Its exasperating. I understand granularity in most cells is not a good sign. Does this apply to a macrophage line as well? Please describe your raw264.7 cells, or if possible post a picture so I can compare with mine.
Using trypan blue and counting, my cells have a viability of ~80-90% the immediate day after thawing. The cells look the most healthy at this stage (to my untrained eye), although they are mostly circular and not completely adherent yet. The following day, after a media change, they look scary again (see above description) and viability can drop to as low as ~60% in just 24 hours
Here are the conditions I use; perhaps you can give me some input:
1. Growth Media - 90% DMEM (4.5g/L glucose, 4mM glutamine, 1.5g/L sodium bicarbonate) + 10% non heat inactivated FBS. I constitute the DMEM powder in autoclaved distilled H20, add the FBS, then filter through a 0.22 micron filter.
2. Passaging - remove old media, rinse with 1XPBS, add some fresh media, scrape (as per ATCC instructions) , resuspend the cell mixture and seed in new flask/s. I split every 2-3 days (about 80% confluence).
3. Freeze - 70% DMEM + 20% FBS + 10% DMSO. I make this fresh when i freeze cells. Because of the small volume required, i dont filter the freeze medium. Is this something to be concerned with? The freezing procedure I use is -20C for 1 hour in a Stratagene cooler box, -80C for 24 hours and then to Liquid N tank.
4. Thawing - rapid thaw in 37C waterbath and immediately transfer to culture flash with pre-warmed complete medium. I dont spin down. Next day replace medium or split (i tend to have a high cell count).
Im need determine what's wrong with my cells, or possibly the cell culture environment. I have some ideas:
1. it may be that at time of freezing (back in July), i didnt do a good job of it to start with so now they are like that. I also did not take not of the morphology enough at that time to remember if they were already like that (my fault).
2. My cells are grown without antibiotics, and are not contaminated with bacteria or yeast. I havent ruled out a mycoplasma infection yet though as we are still waiting for a commercial kit to arrive.
3. Im also suspecting possibility of LPS (endotoxin) somewhere which according to reports is detrimental to macrophage growth but is difficult to detect apparently. If I have never encountered a bacterial infection in my cells, can I rule out LPS contamination? HAs anyone used the HEK-Blue LPS Detection Kit by invivogen?
Any help is much appreciated. Thanks for reading
I think you may tried to add back some old media to the cell (say10% of the total volume) every time you passage the cell. Someone told me that some cell may produce some grow factor that assist their growing. If you remove all the old media and put the new media, the cell may not grow properly.
When freezing the cell use half old media and half new media and a bit of DMSO.
I hope this may help.
50ml FCS and 5ml of L-glute in 500ml of RPMI to culture RAW cells. When we freze it down we use 90% FCS and 10%DMSO and very similar procedures to you guys. You will find that it is normal for some of the cells to start going senile ) long and irrugular shaped after many passages around about 15-20 for us.