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Passage Procedure for RAW 264.

7 Cells
AfCS Procedure Protocol PP00000159 Version 1, 08/20/03

RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice. RAW 264.7 cells used by the Alliance for Cellular Signaling (AfCS) were obtained from the American Type Culture Collection (ATCC; cat. no. TIB-71; lot no. 2263775), expanded, and stored in aliquots for use by AfCS laboratories (Preparation of Frozen Stocks of RAW 264.7 Cells, PP00000180). Stock vials of frozen AfCS cells are thawed using the Thaw Procedure for RAW 264.7 Cells (PP00000160) and then maintained in RAW 264.7 growth medium 1 (RAWGM1) at 37 C in a humidified atmosphere with 5% CO2. For routine maintenance in culture (passage), cells are seeded at a confluence of approximately 10% (1 x 106 and 3 x 106 cells in 100mm and 150-mm plates, respectively) and grown to a confluence of approximately 80%. This procedure requires the cells to be split every two days. For passage over the weekend, 3.3 x 105 and 7.5 x 105 cells are seeded in 100mm and 150-mm dishes, respectively, to ensure no more than 80% confluence when harvested after three days. Cultures are not maintained beyond three months. New cultures are thawed (PP00000160) at least two weeks prior to end of the three-month culture period. Murine macrophages and macrophagelike cell lines such as RAW 264.7 adhere to tissue culturegrade plastic through cationdependent integrin receptors and other cation-independent receptors, predominantly the murine scavenger receptors (MSRs) (Fraser, Hughes, and Gordon. [1993] Nature 364[6435]:343-346). In order to reduce adhesion during routine culture, the RAW 264.7 cells are grown on sterile non-tissuegrade plastic (ultra-dish Petri dishes). Adherence of macrophages to these plates is mediated by M2 (CR3) integrins (Rosen and Gordon. [1987] J. Exp. Med.166[6]:1685-1701), and this interaction is readily reversed by using cation chelators such as EDTA. This adhesive interaction is also sufficiently weak that cells may be detached by the sheer force of media flowing over the cells. Note: macrophages are extremely sensitive to lipopolysaccharide (LPS) endotoxin from Gram-negative bacteria. LPS has major effects on macrophage phenotype and function, including adhesion. All solutions, buffers, and media should be made with sterile, endotoxin-tested, distilled, deionized water. Passage Procedure 1. Transfer RAW 264.7 growth medium 1 (RAWGM1) to a T150 flask (100 ml of growth medium per flask). 2. Place flask in incubator at 37 C with a 5% CO2 atmosphere for at least 30 min to equilibrate the medium. (Note: if the medium is clearly pink and likely has a pH >7.6, remake the medium; all manipulations and additions to cells are done with sterile tissue culture technique.) 3. Enter the bar code of the cells into the cell line GUI. 4. Remove Petri dishes containing cultured RAW 264.7 cells from the incubator. Gently pass a cell lifter over the surface of the dish to dislodge

5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15.

16. 17. 18. 19.

any adherent cells into the medium. Prior to transferring the cell suspension, collect all possible cells by tipping the Petri dish at a 20degree angle and gently streaming the medium over the cells using a 10ml pipette. Avoid creating bubbles by not taking up and expelling air with medium in the pipettes. Several dishes of cultured cells may be pooled for counting. Transfer the single cell suspension to the vessel containing the medium originally removed from the Petri dish. Centrifuge the tube of cells at 400 x g for 5 min at 25 C. Aspirate the medium. Add a volume of equilibrated, fresh growth medium (1/3 the volume of the culture volume). Gently pipette up and down to create a single cell suspension using a 10ml pipette. Remove a volume of the single cell suspension and dilute with trypan blue solution and magnetic cell sorting buffer (MACs buffer) for counting. For example, remove 10 l of the single cell suspension and transfer to a microfuge tube containing 90 l of MACs buffer and 100 l of trypan blue solution. Gently mix the 1 to 20 dilution of cells by pipetting. Count the dilution of white and blue cells using a hemacytometer. Enter the counts into the cell line GUI that is used to calculate the number of cells and the volume needed to seed new vessels. Add the volume of equilibrated RAWGM1, calculated by the cell line GUI, to a new vessel (determined to yield 10 ml/100 mm and 30 ml/150 mm). Pipette the cell suspension up and down, without introducing bubbles, to resuspend the settled cells. Transfer the volume of RAW 264.7 cells, calculated by the cell line GUI (to yield 1 x 106 total cells/100 mm and 3 x 106 total cells/150 mm) to the new dish. Pipette up and down gently 2 to 4 times to mix and evenly distribute the cells in the dish. Place the dish carefully in a humidified incubator set at 37 C with an atmosphere containing 5% CO2. Steps 13 through 16 can be repeated to seed multiple dishes. Aspirate any residual suspended cells for disposal.

Reagents and Materials RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol ID PS00000510 Ultra-dish Petri dish (standard) 60 x 15 mm: Midwest Scientific; catalog no. 901 Ultra-dish Petri dish (standard) 100 x 15 mm: Midwest Scientific; catalog no. 900 Ultra-dish Petri dish (standard) 150 x 15 mm: Midwest Scientific; catalog no. 902

Passage Procedure for RAW 264.7 Cells AfCS Procedure Protocol PP00000159

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Cell lifter: Corning Inc., Costar; catalog no. 3008 Trypan blue solution: Sigma-Aldrich; catalog no. T8154 Magnetic cell sorting buffer (MACS buffer): AfCS Solution Protocol ID PS00000001 Hemacytometer: Fisher Scientific; catalog no. 02-671-5

Author: Robert Hsueh and Tamara Roach Date: 08/20/03 Approved: Paul Sternweis

Passage Procedure for RAW 264.7 Cells AfCS Procedure Protocol PP00000159

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