SCC#10細(xì)胞

年限： embryo, blastocyst

運(yùn)輸方式： 凍存運(yùn)輸

器官來源： 胚胎

細(xì)胞類型： 其他細(xì)胞類型

是否是腫瘤細(xì)胞： 0

物種來源： 小鼠

數(shù)量： 大量

品系： 129X1/SvJ

ATCC Number： SCRC-1020?

組織來源： Inner Cell Mass

細(xì)胞形態(tài)： 造血干細(xì)胞

Designations: SCC#10

Depositors: TJ Ley

Biosafety Level: 1

Shipped: frozen

Medium & Serum: SCC#10細(xì)胞See Propagation

Organism: Mus musculus

Morphology: stem cell

Source: Organ: embryo

Cell Type: stem cell embryonic stem cell;

Strain:129X1/SvJ

Tissue: Inner Cell Mass

Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Restrictions: Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact Washington University in St. Louis' Office of Technology Management Development at kratochj@wustl.edu or 314-747-0923.

Isolation: Isolation date: 2003

Applications: The 129Sv/J ES line was derived from a 129Sv/J (129X1Sv/J in the current nomenclature) strain of mouse, allowing for production of knock-out and transgenic mice.

Cytogenetic Analysis: 40 XY, diploid

Age: embryo, blastocyst

Gender: male

Comments: SCC#10細(xì)胞This mouse ES cell line has been shown to be germline competent. The 129Sv/J ES line was derived from a 129Sv/J (129X1Sv/J in the current nomenclature) strain of mouse, allowing for production of knock-out and transgenic mice. 129Sv/J is a parental substrain of 129 strains and has a white-bellied, light chinchilla or albino coat color with pink eyed appearance. This strain is homozygous for Cdh23ahl, the age-related hearing loss 1 mutation, resulting in progressive hearing loss with onset prior to 3 months of age.

Propagation: ATCC complete growth medium: Mouse ES Cell Basal Medium

Temperature: 37.0°C

Growth condition: feeder cells required.

Subculturing: Protocol: Establishing and maintaining your culture: To insure the highest level of viability, be sure to warm media to 37°C before using it on the cells.

Plate mitotically arrested mouse embryonic fibroblasts (MEFs: CF-1 (ATCC SCRC-1040) as a feeder layer at approximately 55,000 feeder cells/cm2 in complete medium for feeder cells. Refer to the product sheet for mitotically arrested MEFs (ATCC SCRC-1040.1 or ATCC SCRC-1040.2a) for detailed handling instructions. One hour before thawing the vial of ES cells, perform a 100% medium change using complete growth medium for ES cells.

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. Thawing should be rapid (approximately 90 seconds).

Remove the vial from the water bath before the contents are completely thawed, and decontaminate by dipping in or spraying with 70% ethanol. SCC#10細(xì)胞All of the operations from this point on should be carried out under strict aseptic conditions.

Transfer the vial contents plus 5 mL of complete growth medium for ES cells to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete growth medium for ES cells to bring the total volume to 10 mL.

Spin the cells at 270 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 2 mL of complete growth medium for ES cells.

Add the 2 mL of cell suspension to the appropriate size flask containing feeder cells and fresh complete growth medium (see batch specific information). ES cells should be plated at a density of 30,000 to 50,000 cells/ cm2.

Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1 to 2 days.

Subculturing Procedure: To insure the highest level of viability, be sure to warm media and Trypsin - EDTA to 37°C before using it on the cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 every 1 to 2 days is recommended. Plating densities should range from 30,000 - 50,000 cells/ cm2. Note: If the colonies are close to or touching each other the culture is overgrown . Overgrowth will result in differentiation.

Prepare enough flasks with MEFs as stated above in step #1.

Aspirate the medium from the flask(s) with the ES cells.

Wash with PBS (Ca+2/Mg+2-free, ATCC SCRR-2201).

Add 3.0 mL of 0.25% (w/v) Trypsin - 0.53 mM EDTA solution (ATCC 30-2101) to each T75 and place the flasks in the incubator. After one minute the ES colonies will dissociate and the cells will begin to detach from the flask.

Dislodge the cells by gently tapping the side of the flask then wash the cells off with 10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.

Transfer the cell suspension to the appropriate size centrifuge tube.

Spin the cells at 270 x g for 5 min. Aspirate the supernatant.

Resuspend in 30 to 50 mL of fresh culture medium, depending on the split ratio.

Aspirate the medium from flasks containing feeders and replace it with cell suspension (15 mL/flask).

Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1 to 2 days.

medium Renewal: Every day.

subcultivation Ratio: SCC#10細(xì)胞A subcultivation ratio of 1:4 to 1:7 is recommended.

Preservation: Storage temperature: liquid nitrogen vapor phase

Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.

References: 23296: Gossler A, et al. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA 83: 9065-9069, 1986. PubMed: 3024164

16173130: Festing MF, et al. Revised nomenclature for strain 129 mice. Mamm. Genome. 10(8):836, 1999.[PubMed: 10430671]

16173132: Stevens LC. A new inbred subline of mice (129-terSv) with a high incidence of spontaneous congenital testicular teratomas. J. Natl. Cancer. Inst. 50(1): 235-242, 1973. PubMed: 4692863

16173166: Simpson EM, et al. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16(1):19-27, 1997. PubMed: 9140391

16173171: Threadgill DW, et al. Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm. Genome. 8(6):390-393 1997. PubMed: 9166580