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Can't culture RAW 264.7 well? These tips are ignored.

Source: PricellaPublished: 2023-02-10

People who have cultured the RAW 264.7 cell line are likely to be troubled by a problem: the cells are too easy to differentiate! How can we culture them well? What are the tips for RAW 264.7 cell culture? Today we would like to share some experience with you.


Elementary tips

The first step in RAW 264.7 cell culture is to have a thorough understanding of its basic culture conditions, such as growth characteristics, cell morphology, required medium, culture environment, passage ratio and so on.

 

Cell Name

RAW 264.7 Cell Line (Mouse mononuclear macrophage leukemia cell)

Synonym

RAW264; RAW2647; RAW 264.7; RAW-264.7; Raw 264.7; RAW 264.7

Species

Mouse (male, adult BALB/c)

Tissue

Abelson murine leukemia virus-induced tumors

Morphology

Irregular form (round / short spindle-shaped)

Growth Properties

Adherent

Culture Conditions

Atmosphere: Air, 95%; CO2, 5%

Temperature: 37°C

Split Ratio

1:3 - 1:6

Complete Growth Medium

DMEM (PM150210)+10% FBS+1% P/S (PB180120)

Cryopreservation

55% DMEM + 40% FBS + 5% DMSO

          

 

                                                                          

                                                                                               Cell growth of RAW 264.7

 

Advanced tips_1

In addition to basic conditions, other external factors are also likely to cause the "temper tantrum" of the cell, such as bumps in transportation, improper passage methods and so on. In view of several common problems, I would like to share with you how to deal.

 

Problem 1: Can’t find the cells when receiving the package

We got this kind of feedback many times: after receiving the cells, the customer opened the package with full expectations, but can't find the cells under the microscope! This is because RAW 264.7 cells were loosely attached to the wall and may fall off due to bumps during transportation. The detached cells suspended in the medium were not easy to be observed.

Don't worry. Most of the time we can see the cells after putting them still in the incubator for two hours. If only a few cells are seen after standing still, centrifugation verification is the best method at this time. Please transfer all the medium to a centrifuge tube, and centrifuge at 1200 rpm (about 250g) for 3 minutes. Usually, the cell pellet can be observed in this way.

 

Problem 2: cells do not adhere to the wall

After collecting the cells by centrifugation and re-suspended them with a fresh medium in a new flask, the cells may float in clumps and not stick to the wall. In this case, it’s suggested to re-collect the cells and re-suspend them in a 1mL medium. Next, disperse the cells by pipetting slowly and gently, until the cells are separated into single cells. Then we can re-seed the cells.

RAW 264.7 tends to clump after shedding. Cells are alive when clump, and it is difficult to stick to the wall at this time. Be sure to disperse the clumped cells apart into individual cells, otherwise, the cells will be hard to adhere to the wall.


Advanced tips_2

The correct passage method is the key to RAW 264.7 cell culture. We have to be very careful with every step because the cells will differentiate if any notice is neglected. RAW 264.7 cells cannot be digested by trypsin, and many researchers adopt the cell scraping method, which is a very classic method. Pricalla has figured out a better way during years of culturing: pipetting method. The operation is simple, which is friendly to the new operators and can better control the differentiation rate.

 

Passage steps:

a.Give the cell culture flask a few taps.

b.Flush the cells down the flask wall by pipetting with a 1ml pipette or Pasteur pipette (discard those that cannot be flushed off).

c.Transfer the detached cells to a 15ml centrifuge tube.

d.Centrifuge at 1200rpm (about 250g) for 3min.

e.Remove supernatant and re-suspend cells with fresh medium. Proportionally seed the cells to a new flask.

 

Timing for passage:

a.When the cell density is low, it may not be easy to flush the cells down.

b.When the cell density reaches 80%~90%, it is the best time to flush the cells down.                           

                                                                       

                                                                  Recommended cell density for RAW 264.7 passage

 

Hi-end tips

 We've talked a lot about how to deal with the differentiation of RAW 264.7 macrophages, but what is the morphology of the cells that are differentiated? The differentiated cells are polygonal and significantly enlarged, often with long antennae.