器官來源： 胚胎

數量： 大量

運輸方式： 凍存運輸

生長狀態： 貼壁生長

年限： embryo

細胞類型： 成纖維細胞

品系： SIM (Sandos Inbred Mice)

ATCC Number： SCRC-1049?

細胞形態： 成纖維樣

是否是腫瘤細胞： 0

物種來源： 小鼠

Designations: SNL76/7

SNL76/7細胞Depositors: A Bradley

Biosafety Level: 1

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Mus musculus

Morphology: fibroblast

Source: Organ: embryo

Strain SIM (Sandos Inbred Mice)

Cell Type: fibroblast

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Isolation: Isolation date: 1988

Applications: SNL76/7細胞Note: ATCC tested that this cell line is resistant to G 418 (neomycin): 350 ?g/ml.

SNL76/7 was clonally-derived from a STO cell line that expresses both G418 resistance and leukaemic inhibitory factor (LIF) at an abundant level.

This cell line can be used as a feeder layer to support the growth of mouse embryonic stem cells and induced pluripotent stem (iPS) cells.

Age: embryo

Gender: male and female mixed

Comments: SNL76/7 was clonally-derived from a STO cell line that expresses both G418 resistance and leukaemic inhibitory factor (LIF) at an abundant level.

The STO cell line was transfected with a G418-resistance cassette, RV4.0 and a LIF expression construct.

This cell line can be used as a feeder layer to support the growth of mouse embryonic stem cells and induced pluripotent stem (iPS) cells.

Inclusion of G418 in the medium is not necessary for normal cell growth.

Note: ATCC tested that this cell line is resistant to G 418 (neomycin): 350 ?g/ml.

Propagation: ATCC complete growth medium: The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

Temperature: 37.0°C

Atmosphere: air, 95%; carbon dioxideSNL76/7細胞 (CO2), 5%

Subculturing: Establishing culture from frozen cells: To insure the highest level of viability, be sure to warm media to 37?C before using it on the cells. Flasks do not need to be coated before plating MEFs.

Thaw the vial by gentle agitation in a 37?C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.

Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

Transfer the vial's contents plus 5 ml of complete medium (see ATCC complete growth medium for recipe) to a 15 ml centrifuge tube. Use an additional 1 ml of medium to rinse the vial and transfer the liquid to the 15 ml tube. Add 4 ml of complete medium to bring the total volume to 10 ml.

Gently mix and pellet the cells by centrifugation @ 270x g for 5 minutes.

Discard the supernatant and resuspend the cells with 10ml fresh growth medium (warm) and plate cells at seed density of 0.8 X 104 cells/cm2.

Add 5 ml more fresh growth medium (warm) to flask.

Incubate 37?C in a 5% CO2 in air atmosphere.

Fluid change twice a week or when pH decreases. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes. to allow the medium to reach its normal pH (7.0 to 7.6).

Subculturing Procedure:

Cells should be split before they reach confluency.

To insure the highest level of viability, warm culture medium, PBS and Trypsin-EDTA to 37.0°C before use.

Volumes used in this protocol are for 75 sq. cm. flasks; proportionally reduce or increase reagent volumes for culture flasks of other sizes.

Remove and discard culture medium.

Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.

SNL76/7細胞Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37C to facilitate dispersal.

Add 2.0 to 3.0 ml of complete growth medium and aspirate cells by gently pipetting.

Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of approximately 6 X10(3) cells/sq. cm.is recommended.

Incubate cultures at 37.0°C in a 5% CO2 in air atmosphere.

Subcultivation Ratio: 1:10

Interval: every 3 days

Preservation: Freeze medium: complete growth medium supplemented with an additional 35% fetal bovine serum and 10% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

Related Products: Derivative: ATCC SCRC-1050

Recommended medium (without the additional serum described under ATCC Medium): ATCC 30-2002

Recommended serum: ATCC 30-2020

0.25% (w/v) Trypsin - 0.53mM EDTA in Hank's BSS (w/o Ca++, Mg ++): ATCC 30-2101

Cell culture tested DMSO: ATCC 4-X

Phosphate-buffered saline: ATCC 30-2200

References: 22928: Williams RL, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684-687, 1988. PubMed: 3143916

16173313: McMahon AP, Bradley A. SNL76/7細胞The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 62(6): 1073-1085, 1990. PubMed: 2205396

16173314: Takahashi K, et al. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2(12): 3081-3089, 2007. PubMed: 18079707

16173315: Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51(3): 503-512, 1987. PubMed: 2822260

16173317: Okita K, et al, Generation of germline-competent induced pluripotent stem cells. Nature 448(7151): 313-317, 2007. PubMed: 17554338