Hello

Usually RAW264. 7 have a rounded morphology. On stimulation the morphology changes to somewhat polygonal or elongated with protrusions.

Regarding the morphology you can visit ATCC website.

In the images I can see that the cells are sparsely distributed. You need to wait till the cells become 80% confluent. Only then you may get a decent pellet after centrifugation.

What is the culture medium you are using? DMEM with 10%FBS is generally used to culture RAW 264.7 cells.

Best Wishes.

Hi Fauzia,

generally you want your RAW cells to look "fibroblast-like" in shape and rather small (nicely visualized here: https://www.lgcstandards-atcc.org/products/all/tib-71.aspx?geo_country=de#characteristics)

Some questions that might help to identify the problem:

• At what time-point after seeding did you take the pictures?
• What medium are you using?
• What culture-/platic-ware are you using?
• Did you thaw cryoconserved cells (if yes, how?) or did you get them in culture from someone?
• What passage are the cells at?
• Can you verify that your cells are viable? (e.g. with trypanblue or other methods). Because especially on the first pictures the supposed cell morphology looks quite troubling.

Difficulties obtaining decent pellets after centrifugation is (to a certain degree) "to be expected", as the RAW cells are very small - but with sufficient amounts of cells pellets should be easily visible.

Best regards,

Felix

Dear Malcolm Nobre

Thank you for your reply.

Yes, I am using DMEM +L-glutamine with 10% FBS. I will wait until the culture becomes more confluent. Hopefully, I will get a decent pellet.

Please do let me know if you have any suggestion or tips!

Thank you!

Hi Felix Stegmann ,

Actually, these pictures are from the same plate and the same region, only differ in microscope's focuses and yet resulting in somewhat different morphology. that is my concern... I can verify that the cells are viable (>80%) of viability.

The pictures were taken

Hi Fauzia Izzati ,

thanks for the additional infos. Now i get your initial comment about the different focuses relating to the pictures, I think this explains the differences that you see :)

RAW cells will become adherent after a while (thats why I asked about the time-point after seeding). What you observe are most likely viable cells that begin to attach to the bottom after approx. 24h (the round cells in pictures 3 to 5) while debris will float above it (most likely pictures 1 to 2, also a little bit in the other pictures). Give them some more time to attach; then it should also be way easier to focus them correctly. Especially after thawing cells generally take more time to really adjust.

The medium should be just fine (as Malcolm Nobre also mentioned).

I can't give you an exact cell number regarding the pellet at the moment, but in my hands a confluent T25 flask of cells results in a really large pellet - so even lower amounts should be easily visible.

I hope this helps :)

Best regards

Felix

Thank you so much Felix Stegmann for your reply!

I really appreciate the information from you and they are definitely helpful for me. Well I guess, I have to be patient and see how the culture goes :)

Thanks again

The morphology see in the pics is quite unusual, indicating insufficient cell number or cell death or stressed cells.

Basically, they are look round ball like at the time of seeding and in an hour will adhere to the surface and continue to grow showing slight projections

RAW264.7 cells can grow well in both DMEM and RPMI 1640 media containing 10% FBS (heat inactivated), 2 mM L-glutamine, 100 U of penicillin/ml, and 100 μg ml of streptomycin. The average doubling time of RAW cells is 15 hours. If thats not observed, you can expect something went wrong. I see that the cell number is too low. Cell need to communicate for better growth. You may try 1:3 or 1:4 dilution of cell culture to fresh medium to attain confluence. Moreover, these cells should never be completely confluent. Procedures during subculturing defines the health of the cells. I see a lot of cell death from the images.

While dislodging cells from the flask, press the scraper gently and make only one pass with the cell scraper from the bottom of the flask to the top to prevent excessive damage to the cells. Lifting these cells can be tricky and use of trypsin EDTA helps. However, excessive treatment may induce cell death.

Cells in the images looks heavily stressed !!

Hello Vinod Nadella

Thank you for your answer and explanation particularly about the cell scraper.

I think I might be slightly excessive with the scraper hence the stress.

I am using PBS and cell scraper only.

Do you use trypsin after/before the scraper?

Can you explain more about using trypsin EDTA? In which percentage and how long do you exposed the culture to the trypsin?

Any suggestions from you will be really appreciated

Thank you!

After removing the spent culture media and giving a gently wash with PBS to remove any debris, you can add 1ml of 0.05% trypsin-EDTA and make sure to evenly spread the medium across the culture dish. Incubate at 37C for 2-3 minutes. After incubation, giving a tap on the edges of the dish should get the cell off into the medium. Or, gently scrap. Scrapping generally damage the cells. So, using a cell lifter to dislodge will be ideal. Don't cell the cells sit in trypsin-EDTA for extended time. Once cells are dislodged, add 10ml of fresh culture medium and spin down the cells. To the cell pellet, add fresh media and then sub-culture.

Dear Vinod Nadella

thank you so much for the explanation.

I will try using low concentration of trypsin like you mention. and will see if it works better for me.

if you have any suggestion, i would be grateful!

thank you again

Share your results and if you still encounter with issue, we can dissect where things are going wrong in detail.

But, I hope everything should be fine for you now

BW

Thank you so much Vinod Nadella , I really appreciate it

Hi, Fauzia Izzati,i think your raw cells may be apoptosis。 I detached my raw cells with medium under UV exposure， raw cells looked smaller and rounder， un-adherent，nuclear enrichment，they didn't growth at all for 72h。

Zhang Wen thank you for your reply! yes, my cells were dying. After thawing, the viability was okay but it kept decreasing as I went on with the culture.

After I traced back, the issue might be started since the cells arrived in my lab - I was not present at the time.

I decided to purchase a new vial from the ECACC. The cells from this batch are healthy and I am able to use them in my assays.

Thank you all for your kind help Vinod Nadella Felix Stegmann Malcolm Nobre Zhang Wen