I would really appreciate it if anybody could help me with my problem.
I’ve been working on the macrophage Raw 264.7 cell line for 5 months. Despite following all the advice from ATCC and other guides, I still haven’t succeeded in growing cells beyond the 3rd or 4th subculture. After the second passage, the cells start to fall apart and become debris floating in the medium. Also, some of the attached cells exhibit weird morphology—they appear elongated and branched in different aspects. I’ve uploaded a picture of the cells; they are from the 2nd passage, taken 24 hours after dislodging them from the flask.
I’ve tried both a cell scraper and trypsin-EDTA 0.25% (1X) for dislodging the cells. Additionally, I prepare fresh medium (RPMI, FBS, PBS, etc.) for each use. I’ve also checked for cell culture contamination in the incubator, but other cell lines grow normally except for mine.
Hello Elahe,
That looks like apoptotic cells to me (the ones that start to "fall apart").
Have you used DMEM and does your medium contain L-Glutamine?
These cells also do not like conditions that are too alkaline, i.e. you should make sure that the color of the medium is red to orange rather than pink.
Ute Liegibel Thank you so much for your help. I have used RPMI as a culture medium with 10 % FBS and 1% Pen Strep.
The color of medium is somehow pink when I renew the culture medium, how can I avoid an alkaline condition?
If you use TC flasks with filter caps or grow your cells in petri dishes, gas exchange is guaranteed and a neutral pH value is established. If using cell culture flasks w/o filter cap, unscrew the cap by half a turn to allow sufficient ventilation. Otherwise an alkaline pH value will result.
Elahe Haghighi there are several different methods, the Mycoalert assay by Lonza is good but it's based on luminescence, you will need a luminometer or multimode reader. I know there are assays based on absorbance but I can't remember the name of the assay. Alternatives are PCR using mycoplasma-specific primers and fluorescent probes to check under the microscope.
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