Clear polystyrene flat-bottom, tissue culture-treated multiwell plate with lid. Sterile tissue culture-treated dish; 35 mm, 60 mm, or mm formats. This product is designed for use in the following research area s as part of the highlighted workflow stage s.
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Learn more. Cell Culture Flask. Format Choose an Option Size 50 Flasks. Size 40 Flasks. From: EUR. Add to Cart. Skip to the end of the images gallery. Cell Biologics' products can be purchased through Fisher Scientic by searching Cell Biologics' product catalog number or key words Aged 78 Weeks Mouse Cells. General Protocol for the culture of Primary Fibroblasts. All cell culture procedures must be conducted in a bio-safety cabinet. Any and all media, supplements, and reagents must be sterilized by filtration through a 0.
Use aseptic technique to prevent microbial contamination. Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival. Review the information provided on the Cell Biologics website about appropriate culture media e. Coating of flasks or dishes:. Cell recovery from cryovial:. Expansion of cultured mouse primary cells:. We recommend splitting primary cells at the follow ratio:. Procedure for Freezing Cells.
We recommend freezing primary cells at the follow ratio:. Primary cells differ from cell lines in many ways. Newsletter Register Log In Cart 0. All Categories. Gene Expression and Regulation-Customizing Service Construction of expression plasmids in bacterial and mammalian cells Cloning of functional genes and promoters Prediction and analysis of gene regulation Preparation of expression adenovirus and lentivirus Tissue RNA membranes for northern blot Various mutations of expression plasmids and promoters.
Shopping Cart. Contact Us Site Map. K Aged 78 Weeks Mouse Cells. Culture Fibroblasts General Protocol for the culture of Primary Fibroblasts All cell culture procedures must be conducted in a bio-safety cabinet.
Medium: Review the information provided on the Cell Biologics website about appropriate culture media e. Transfer cells from the vial to a sterile centrifuge tube. Flush the vial with an additional 0. Centrifuge cells at g for 5 minutes.
Also change culture media the following day to remove non-adherent cells and replenish nutrients. Cells should be checked daily under a microscope to verify appropriate cell morphology. Expansion of cultured mouse primary cells: Flush the adherent layer with a 5 ml sterile pipette 3 times to dislodge loosely attached cells.
Remove and discard the cell culture media from the flask. Wash adherent cells 2 times with 10 ml of sterile PBS 1X without calcium and magnesium to remove nonadherent cells or fraction. Remove and discard the wash solution from the flask. Use 2. Cells should be checked daily under a microscopy to verify appropriate cell morphology. Change culture medium every hours. Pre-wash cells with 1X PBS 2 times whenever replacing the medium. We recommend splitting primary cells at the follow ratio: The recommended split ratio for primary murine cells is A confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.
Wash adherent cells times with 10 ml of sterile PBS 1X without calcium and magnesium to Remove nonadherent cells or fraction. The growth of these adherent cells must be closely monitored to ensure cell health.
Bear in mind that cell lines retain many characteristics of the original cell culture, but with each successive passage they can also begin to acquire characteristics unique to the expanded culture. Therefore, consider limiting the number of times you continue to passage an individual cell culture. When passaging cells, it is important to use sterile technique and the appropriate reagents and equipment.
It is essential to use the appropriate media for the optimal growth and expansion of your cell line. Each cell line will require a specific growth supplement cocktail, however, most cell lines at minimum require supplementation with the following: Serum, such as Fetal Bovine Serum, which must be heat-treated to inactivate the bovine complement and which provides vital growth factors for the cells, antibiotics, like penicillin and streptomycin, which help limit contaminating growth in the culture, and other growth factors, like fibroblast growth factor, to help prolong the growth and expansion of the cell lines.
First, take note of the color of the tissue culture media. Fresh, cell culture media is rich in nutrients and appears a clear, orange color, partly due to the addition of the pH indicator, phenol red. As cells begin to use up nutrients in the media, waste and acid begin to build up in the cell culture, lowering the pH.
For this reason, many tissue culture medias contain phenol red, which turns the media from orange to yellow when cells turn the culture acidic. Change the media before it turns this color! When the phenol red turns the media a pinkish color, indicating that the pH has become too basic for healthy cell growth, it may be time to change your CO2 tank as well as your media.
Most adherent cell lines attach naturally to uncoated plastic. Therefore plastic cell culture plates, like petri dishes or 6 well plates, are frequently used for subculturing cells. Plastic cell culture flasks are also used. The T25 cell culture flask, for example, is often used to expand small cell line populations or to start the growth of a slowly expanding cell line.
T75 culture flasks are commonly used for cell lines that proliferate more quickly or to generate larger numbers of cells than can fit in a T25 flask. When removing adherent cells, the proteins that bind the cells to the plastic must first be cleaved. For this purpose, the digestive enzyme trypsin is frequently used.
It is important to carefully time the trypsin exposure, as treatment for too long can result in damage to other cell surface proteins. Cell culture media can contain trypsin neutralizers. Therefore, phosphate buffered saline, or PBS, is often used to wash the cells before trypsinization. Before you begin, it is important to understand how frequently your cells should be monitored, which will depend on how fast your cells proliferate.
Now that your media is a happy orange color, observe the other culture conditions, such as whether or not the culture is cloudy — possibly indicating contamination -- the size and density of the cell colonies, and the overall quality of the cells. Now add trypsin to the cells and then incubate them at 37 C. After about 5 minutes, confirm that the cells have detached, and then stop the proteolysis by adding fresh tissue culture media.
Transfer the cell suspension into a conical tube for centrifugation. Then, after spinning down the cells, carefully remove the supernatant without disturbing the pellet and resuspend the cells in fresh media.
As mentioned earlier, human embryonic stem cells are a type of cell that must be passaged. They are typically cocultured with mouse embryonic fibroblasts, or MEFs, which provide factors that help stem cells retain their pluripotent state. They can be selected by micropipette or by gentle scraping with a glass picking tool and then plated in a new culture for expansion. A cell scraper can be used to gently remove the cells from the bottom of the culture plate.
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