Passage of RAW 264.

7 Cells for FXM Experiments

AfCS Procedure Protocol PP00000226 Version 1, 04/06/04

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, frozen, and distributed to all participating laboratories. The FXM project within the AfCS utilizes a small subset of ligands for stimulation of measured responses affecting intracellular Ca2+ and phosphatidylinositol metabolism including C5a, UDP/UTP, and aggregated IgG2a antibody. FXM RAW cell culture conditions are identical to that of non-FXM studies except that EDTA is used routinely in the harvest procedure and non heat-inactivated fetal bovine serum is used in the growth medium. The magnitudes of responses to Fc RI stimulation via aggregation of IgG2a show greater stability over time in culture if nonheat-inactivated serum is used. The cell adhesion to petri dishes and intercellular adhesion observed in suspension is divalent cation-dependent and brief exposure to EDTA serves to loosen both forms of adhesion and yield single-cell suspensions for passage. The cell doubling time is usually about 15 hr and yields approximately a 3-fold increase in cell number per day. Cultures are grown to a confluence of approximately 80%. Culture density, age, growth rate, and so forth are recorded by passage number by the cell line GUI. Cells should not be used for assays unless cell doubling times are relatively constant. 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 for FXM (RGMFXM) to a T75 flask (30 ml of RGMFXM per flask). (Note: all manipulations and additions to cells are done with sterile tissue culture technique in a laminar-flow hood.) 2. Place flask in an incubator at 37 C with a 5% CO2 atmosphere for at least 30 min to equilibrate medium. (Note: if medium is clearly pink and likely has a pH >7.6, remake medium). 4. Remove petri dishes containing cultured cells from incubator. Gently transfer all growth medium out of each dish into a sterile tube using a sterile pipette (this aliquot may contain viable cells). 5. Immediately add 5 ml PBS/EDTA solution at 4 C and gently rock dish to wash entire surface with solution. 6. Place dish in a 4 C refrigerator for 3 to 5 min. 7. Gently dislodge adherent cells from dish by tipping culture dish at a 20degree angle and gently directing a stream of buffer over cells using a transfer pipette. Rotate plate 180 degrees. Repeat this process until all sections of dish are clear of cells. Avoid creating bubbles by not

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9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

taking up and expelling air with buffer in pipette. Transfer single cell suspension to tube containing medium originally removed from culture dish, cap tube, and place on ice. Multiple dishes of similar cultured cells may be pooled for counting. Multiple lines may also be harvested separately and stored on ice for <1 hr. Centrifuge tube of cells at 400 x g for 5 min at 4 C. Aspirate medium and gently tap tube to disrupt cell pellet. Add a volume of equilibrated, fresh RGMFXM (~1/3 of original culture volume). Gently pipette up and down to create a single cell suspension using a transfer pipette. Remove a volume of single cell suspension and dilute with trypan blue solution and magnetic cell sorting buffer (MACs buffer) for counting. For example, remove 10 l of 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 dilution of white and blue cells using a hemacytometer. Enter bar code of cells into cell line GUI. Enter counts into cell line GUI that is used to calculate number of cells and volume needed to seed new vessels. (density of 0.33 to 2 x 106 total cells in 10 to 20 ml medium per 100-mm dish). Add volume of equilibrated growth medium, calculated by cell line GUI, to a new ultra-dish petri dish. Pipette cell suspension up and down, without introducing bubbles, to resuspend settled cells. Transfer volume of RAW 264.7 cells calculated by cell line GUI to new dish. Pipette up and down gently 2 to 4 times to mix and evenly distribute cells in dish. Place 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 for FXM (RGMFXM): AfCS Solution Protocol PS00000636 Phosphate buffered saline, pH 7.5, 1X, with EDTA, 2 mM, low LPS (PBS/EDTA, pH 7.5, low LPS): AfCS Solution Protocol PS00000631 Ultra-dish petri dish (standard) 100 x 15 mm: Midwest Scientific; catalog no. 900 Trypan blue solution: Sigma-Aldrich; catalog no. T8154 Magnetic cell sorting buffer (MACS buffer): AfCS Solution Protocol PS00000001
Passage of RAW 264.7 Cells for FXM Experiments AfCS Procedure Protocol PP00000226

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Hemacytometer: Fisher Scientific; catalog no. 02-671-5

Author: Robert Rebres Date: 04/06/04 Approved: Tamara Roach

Passage of RAW 264.7 Cells for FXM Experiments AfCS Procedure Protocol PP00000226

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