Product Description

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Cell Biology
ATCC® Number: SCRC-1037™    Price: £288.00; €388.00
Designations: G-Olig2
Depositors:  DI Gottlieb
Biosafety Level: 1
Shipped: frozen
Medium & Serum: See Propagation
Growth Properties: adherent
Organism: Mus musculus (mouse)
Morphology: spherical colony
Source: Strain: 129X1/SvJ
Organ: embryo
Tissue: inner cell mass
Cell Type: embryonic stem cell;
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Isolation: Isolation date: August, 2001
Age: embryo, blastocyst
Comments: G-Olig2 cells were designed by the insertion of green fluorescent protein (GFP) into the gene for Olig2, a lineage-specific transcription factor. This insertion permits visualization and physical separation of a subset of living ES-cell-derived neural cells. [PubMed: 12529550] SCRC-1037 is a subclone of deposited cell line.
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: Establishing and maintaining your culture:
To insure the highest level of viability, be sure to warm media to 37ºC before using it on the cells.
  1. Plate mitotically arrested MEF (CF-1) (ATCCSCRC-1040)as a feeder layer at approximately 1.5 to 2.0 X 10(6) cells/T25 at least one day before plating the cells (see product sheet for mitotically arrested MEF for protocol). One hour before thawing the vial of cells, perform a 100% medium change using 4 ml of complete ES-DMEM (see ATCCcomplete growth medium for recipe).
  2. 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).
  3. 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.
  4. 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.
  5. Spin the cells at 270 xg for 5 min. Aspirate the supernatant and resuspend the pellet in 5 ml of complete ES-DMEM.
  6. Add the 5 ml of cell suspension to the T75 flask containing feeder cells and 10 ml complete ES-DMEM.
  7. 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 37ºC 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 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.
  1. Prepare enough flasks with MEFs as stated above in step #1.
  2. Aspirate the medium from the flask(s) with the cells.
  3. Wash with PBS (Ca+2/Mg+2-free, ATCCcat# SCRR-2201).
  4. Add 3.0 ml of 0.25% (w/v) Trypsin/0.53 mM EDTA solution (ATCCcat # 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
  5. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 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.
  6. Spin the cells at 270 xg for 5 min. Aspirate the supernatant.
  7. Resuspend in 30 to 50 ml of fresh culture medium, depending on the split ratio.
  8. Aspirate the medium from 4 to 7 feeder layer flasks and replace it with 15 ml/flask of the cell suspension.
  9. 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.

Interval: Every one to two days
Subcultivation Ratio: A subcutivation 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
References: 57459: Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999
89294: Xian HQ, et al. A subset of ES-cell-derived neural cells marked by gene targeting. Stem Cells 21: 41-49, 2003. PubMed: 12529550
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