7AC5/EYFP細胞

器官來源： 胚胎

數量： 大量

生長狀態： 貼壁生長

ATCC Number： SCRC-1033?

組織來源： inner cell mass

細胞類型： 其他細胞類型

是否是腫瘤細胞： 0

物種來源： 小鼠

年限： embryo, blastocyst

運輸方式： 凍存運輸

Designations: 7AC5/EYFP

Depositors: A Nagy

Biosafety Level: 1

Shipped: frozen

7AC5/EYFP細胞Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Mus musculus

Morphology:

Source: Organ: embryo, blastocyst

Tissue: inner cell mass

Cell Type: stem cell

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.

Applications: SCRC-1033 has been shown to be germline competent, stains positive for pluripotency markers and alkaline phosphatase activity, and survives puromycin selection at concentrations of up to 0.004 mg/ml.

Age: embryo, blastocyst

Gender: male

Comments: 7AC5/EYFP細胞7AC5/EYFP is a yellow fluorescent variant of R1 ES cells (ATCC SCRC-1011). The fluorescent variant was generated by the random integration of EYFP into R1 ES cells using co-electroporation with a circular selectable marker containing vector pPGK Puro. The vector is driven by a CMV immediate early enhancer coupled to the chicken beta-actin promoter and first intron. SCRC-1033 has been shown to be germline competent, stains positive for pluripotency markers and alkaline phosphatase activity, and survives puromycin selection at concentrations of up to 0.004 mg/ml.

Propagation: ATCC complete growth medium: ES-DMEM (ATCC SCRR-2010) supplemented with 2.0 mM L-Alanyl-L-Glutamine (ATCC 30-2115), 0.1 mM non-essential Amino Acids (ATCC 30-2116), 0.1 mM 2-mercaptoethanol (Invitrogen Life Technologies No. 21985), 1000 U/ml mouse leukemia inhibitory factor (LIF) (Chemicon No. ESG1107) and 15% fetal bovine serum (ATCC SCRR-30-2020).

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37.0°C

Growth Conditions: SCRC-1033 should be grown on Mitomycin C treated or irradiated mouse embryonic fibroblasts in the presence of complete growth medium in order to maintain them in an undifferentiated state.

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

Plate mitotically arrested MEF (CF-1) (ATCC SCRC-1040)as a feeder layer at approximately 60,000 feeder cells/cm2 at least one day before plating the cells (see mitotically arrested MEF product sheet for protocol). Check batch specific information for appropriate size flask. One hour before thawing the vial of ES cells, perform a 100% medium change using complete ES-DMEM (see below for recipe).

Thaw the vial by gentle agitation in a 37C 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. 7AC5/EYFP細胞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 ES-DMEM 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 ES-DMEM 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 ES-DMEM.

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 40,000 to 50,000 cells/ cm2.

Incubate the culture at 37C 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 37C 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 1:6 every 1 to 2 days is recommended. Plating densities should range from 3 to 4 X 10(6) cells/ T75. 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 37C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1 to 2 days.

Medium Renewal: 7AC5/EYFP細胞every day

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

Storage temperature: liquid nitrogen vapor phase

Related Products: parental cell line:ATCC SCRC-1011

recommended serum:ATCC SCRR-30-2020

MEM Non-Essential Amino Acid Solution, 100x:ATCC 30-2116

Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC SCRR-2010

L-Alanyl-L-Glutamine Solution, 200 mM:ATCC 30-2115

References: 57459: Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

90551: Hadjantonakis AK, et al. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76: 79-90, 1998. PubMed: 9867352

90552: Hadjantonakis AK, Nagy A. FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 27: 95-98, 2000. PubMed: 10951501

90553: Hadjantonakis AK, Nagy A. 7AC5/EYFP細胞The color of mice: in the light of GFP-variant reporters. Histochem. Cell Biol. 115: 49-58, 2001. PubMed: 11219608

90554: Hadjantonakis AK, et al. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2: 11, 2002. PubMed: 12079497