Culturing RAW 264.7 Cells: Difference between revisions

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Murine macrophages and macrophage–like cell lines such as RAW 264.7 adhere to tissue culture–grade plastic through cation–dependent 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-tissue–grade plastic (ultra-dish Petri dishes). Adherence of macrophages to these plates is mediated by αMβ2 (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==~~

==Reagents and Materials==

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

==Passage Procedure==

#Transfer RAW 264.7 growth medium 1 (RAWGM1) to a T150 flask (100 ml of growth medium per flask).

#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.)

#Enter the bar code of the cells into the cell line GUI.

#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

#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 any adherent cells into the medium. Prior to transferring the cell suspension, collect all possible cells by tipping the Petri dish at a 20 degree angle and gently streaming the medium over the cells using a 10-ml 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.

any adherent cells into the medium. Prior to transferring the cell suspension, collect all possible cells by tipping the Petri dish at a 20-degree angle and gently streaming the medium over the cells using a 10-ml 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.

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#Steps 13 through 16 can be repeated to seed multiple dishes.

#Aspirate any residual suspended cells for disposal.

[[ Category:Cell Culture ]]

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 Murine macrophages and macrophage–like cell lines such as RAW 264.7 adhere to tissue culture–grade plastic through cation–dependent 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-tissue–grade plastic (ultra-dish Petri dishes). Adherence of macrophages to these plates is mediated by αMβ2 (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==
 ==Reagents and Materials==
@@ Line 16: / Line 15: @@
 *Hemacytometer: Fisher Scientific; catalog no. 02-671-5
+==Passage Procedure==
 #Transfer RAW 264.7 growth medium 1 (RAWGM1) to a T150 flask (100 ml of growth medium per flask).
 #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.)
 #Enter the bar code of the cells into the cell line GUI.
-#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
+#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 any adherent cells into the medium. Prior to transferring the cell suspension, collect all possible cells by tipping the Petri dish at a 20 degree angle and gently streaming the medium over the cells using a 10-ml 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.
-any adherent cells into the medium. Prior to transferring the cell suspension, collect all possible cells by tipping the Petri dish at a 20-degree angle and gently streaming the medium over the cells using a 10-ml 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.
@@ Line 36: / Line 35: @@
 #Steps 13 through 16 can be repeated to seed multiple dishes.
 #Aspirate any residual suspended cells for disposal.
+[[ Category:Cell Culture ]]