CE3細胞

器官來源： 胚胎

細胞形態： 其他

細胞類型： 胚胎干細胞

生長狀態： 貼壁生長

數量： 大量

是否是腫瘤細胞： 0

物種來源： 小鼠

年限： embryo, blastocyst

ATCC Number： SCRC-1039?

運輸方式： 凍存運輸

品系： 129S2/SvPas

組織來源： inner cell mass

Designations: CE3

Depositors: DI Gottlieb

CE3細胞Biosafety Level: 1

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Mus musculus

Morphology: sperical colony

Source: Organ: embryo

Strain: 129S2/SvPas

Tissue: inner cell mass

Cell Type: embryonic 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.

Restrictions: CE3細胞Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact ATCC 's Office of Licensing and Business Development at licensing@ATCC .org or 703 365 2773.

Applications: Neural differentiated CE3 cells are intensely GFP+ and suitable for cell tracking in tissue culture and transplantation.

The CE3 cell line was derived from the D3 ES cell line [PubMed: 12591158].

Age: embryo, blastocyst

Comments: CE3 (for Cassette Exchange) contains one 'acceptor' module that allows for efficient double lox targeting, with constitutive GFP expression. Neural differentiated CE3 cells are intensely GFP+ and suitable for cell tracking in tissue culture and transplantation. The cell line is also puromycin resistant. The CE3 cell line was derived from the D3 ES cell line [PubMed: 12591158].

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

Subculturing: Protocol: CE3細胞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 MEF (CF-1) (ATCC SCRC-1040) as a feeder layer at approximately 5.0 to 6.0 X 10(6) cells/T75 at least one day before plating CE3 cells (see product sheet for mitotically arrested MEF for protocol). One hour before thawing the vial of CE3 ES cells, perform a 100% medium change using 10 ml of complete ES-DMEM (see ATCC complete growth medium for recipe).

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 as soon as the contents are thawed, 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 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 xg for 5 min. Aspirate the supernatant and resuspend the pellet in 5 ml of complete ES-DMEM.

Add the 5 ml of cell suspension to the T75 flask containing feeder cells and 10 ml complete ES-DMEM.

Incubate the culture at 37?C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 2 to 3 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 2 to 3 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 . CE3細胞Overgrowth will result in differentiation.

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

Aspirate the medium from the flask(s) with CE3 ES cells.

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

Add 3.0 ml of 0.25% (w/v) Trypsin/0.53 mM EDTA solution (ATCC cat # 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.

Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7 to 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.

Spin the cells at 270 xg 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 4 to 7 feeder layer flasks and replace it with 15 ml/flask of CE3 cell suspension.

Incubate the culture at 37?C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 2 to 3 days.

Interval: Every 2 to 3 days

Subcultivation Ratio: A split ratio of 1:4 to 1:7 is recommended

Medium Renewal: 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: recommended serum: ATCC SCRR-30-2020

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

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

89300: Adams LD, et al. Double lox targeting for neural cell transgenesis. Brain Res. Mol. Brain Res. 110: 220-233, 2003. PubMed: 12591158