By splitting by pipetting, you are in essence selecting for a non-adherent subpopulation. I have seen this with several cell lines, often with phenotypic changes [i.e., loss of surface antigens]. In my lab we carried RAW264 cells for years, but always passaged using a rubber policeman or scraper. I definitely recommend trying that.

Maybe by merely pipetting you inadvertently selected for cells with a suspension phenotype. We work with J774 macrophages which are similar to RAW cells and we scrape the cells with a rubber cell scraper to split them. This works fine for us. I would not trust the cells if they are only "floating" as you say, as RAW cells should have a adherent phenotype.

Using cell scraper to subculture RAW264.7 works well in our lab.

Are the cells fully alive? They can be grown as non-adherent cells if the plates are not tissue culture grade. If you use treated plates, try different batches of serum. Did you test for mycoplasma infection?

The best way of spliting RAW 264.7 is by Scraping them with a Rubber cell scraper..

As for the floating cells better not to use them ,they might be dead.

If you have a new batch of cells you better start with that , and the next day change the medium with a new one ,wait another( 2-3) days until they are confluent culture and then split them as mentioned before .

Usually the floating cells are going to die, you can assess that by trypan blue method. However you collect those floating cells and centrifuge them in a sterile condition then the pellet can be cultured again with little amount of media then you can add a medium gradually to ensure that all cells are attached. Normally cells go to float due to either they are dead cells or over-confluent.

For splitting, you should use scrapper and do it very gently. I think if you avoid trypsin treatment, and follow scraping, then you would be able to reduce cell floating.

In the splitting process, I washed the cells down from the dish by pipetting without touching the cells. And the floating cells' vialibility was assessed by trypan blue and the cells were alive. Maybe as Yannick Waumans' saying, by merely pipetting inadvertently selected for cells with a suspension phenotype.

And can the rubber cell scraper be reused or I have to use a new one at every splitting ?

Thanks for your suggestion.But the RAW cells' nitric oxide (NO) concentration will become high under the stimulation of trypsin.

Have tried using EDTA in combination with trypsin for detaching the cells, if this not useful try to use collagenase ty[e II instead of trypsin because sometimes the trypsin is harmful for cells.

By splitting by pipetting, you are in essence selecting for a non-adherent subpopulation. I have seen this with several cell lines, often with phenotypic changes [i.e., loss of surface antigens]. In my lab we carried RAW264 cells for years, but always passaged using a rubber policeman or scraper. I definitely recommend trying that.

I agree with Gary Gilmore. Here is the recommended culture method by ATCC for this cell line:

"Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

Subculturing

Subcultures are prepared by scraping. For a 75 cm2 flask, remove all but 10 mL culture medium (adjust amount accordingly for other culture vessels). Dislodge cells from the flask substrate with a cell scraper; aspirate and add appropriate aliquots of the cell suspension into new culture vessels.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:6 is recommended

Medium Renewal: Replace or add medium every 2 to 3 days.

Culture Conditions

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37°C"

http://www.atcc.org/Products/All/TIB-71.aspx#7301B7F956944F8382B6192957C08A3B

I concur Gary and others above- always follow ATCC's recommendations for cell culturing unless you have a very specific reason for any deviation. Use a rubberized cell scraper/lifter, and I would suggest using disposable ones to avoid any contamination issues. If you think that trypsin is causing iNOS activation and NO production, you could try another cell harvesting mix like Accutase, which is much gentler, and could be an alternative if the cell scraping also seems to activate the RAWs.

Thank you for everyone's suggestion.I tried the scrapper way and used the RAW cells to do a LPS-induced nitrate oxide expreiment. The nitrate oxide concentration of RAW cells was about 5 µM and the LPS-induced NO concentration was about 55 µM. But the drug which shoud decrease the LPS-induced NO concentration of RAW cells didn't perform the effect. I don't know whether the passage way by scrapper has any influence on the RAW cells.

On the other way, I continuted to split the RAW cells by pippetting , but they are not floating now and becoming adherent in the dish firmly. It's very strange. I don't know why and worried about the influence of different passage way on the cells.

RAW cells are macrophages and when they are activated, they are floating and their morphology is different. You have to pay attention because it's very easy that these cells are activated

Although macrophages usually prefer to grow floating in media but this cell line was adapted to grow adherent to the flask base. Sometime when you grow this cell line for the first time after freezing, most cells appear tired and have no ability to stick down. Moreover sometimes we use wrong culture medium or wrong serum percentage, these will affect the behavior of those cells.

Well maybe I ma late for this question but you can try 4ºC calcium magnesium free PBS-EDTA 2mM . Surface attachment of MO is calcium dependent, so EDTA is the key. I have a lot of experience in RAW cells.

Good luck

Hi everyone, i have a problem with my experiment. I stimulated Raw 264.7 by LPS, and measured NO concentration. The raw 264.7 untreated LPS but released NO (high concentration).

I split the cells by pipetting, Is that a reason?

In vitro floating cells usually represent either dead cells or overgrowing cells. Therefore you have to check their viability by trypan blue. Meanwhile try to remove cells gently during subculturing as some cells are fragile and can damaged easily.

Overgrowing the cell culture activate the cells. A constant repetition of this procedure damage the cell population.

I have received  New Raw cell line from NCCS pune.how to proceed to maintain it

We carry RAW264-7 in RPMI1640/10%heat-inactivated FBS with pen/strep/fungizone; I have also used untreated FBS and used gentamicin SO4 instead of Pen/strep/fungizone. We seed the cells at 2.5 x 10^5 per ml - typically we put 10 ml in a T75 culture flask. Passage by scraping cells with a rubber policeman or scraper every 2-3 days. Typical yields are 1.5 - 2.5 x 10^7 per flask.