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Cell Biology
ATCC® Number: SCRC-1019™    Price: $649.00
Designations: B6/BLU
Depositors:  TJ Ley
Biosafety Level: 1
Shipped: frozen
Medium & Serum: See Propagation
Organism: Mus musculus (mouse)
Morphology: stem cell
Source: Strain: C57BL/6
Organ: embryo
Tissue: inner cell mass
Cell Type: stem cell embryonic stem cell;
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Isolation: Isolation date: 1998
Cytogenetic Analysis: 40 XY, diploid
Age: embryo, blastocyst
Gender: male
Comments: This mouse ES cell line has been shown to be germline competent. The B6/BLU ES cell line was derived from a C57BL/6 transgenic line that contains a LacZ reporter. The transgene is a -globin LacZ fusion expressed exclusively in peripheral red blood cells. The transgene was assembled in pUC19, and contains the 1.9 kb KpnI-PvuII human 54 HS-2 fragment, upstream from the marked ß-globin transgene. The 5' part of the human ß-globin gene was fused to a 3' kb NcoI-BglII fragment obtained from pLacD. The 3' part of the -globin gene consisted of a 2.8 kb BamHI-XbaI fragment that contains the 3' end of exon 2, intron 2, exon 3 and 3' flanking sequence. 5' HS-2 was inserted in the genomic (5' to 3') orientation with respect to the transgene. The transgene was isolated from the plasmid vector backbone by cleavage with XhoI and SalI .Instead of determining chimerism visually by coat color or assessing chimerism genotypically in a tail DNA, chimerism is assessed quantitatively in the mesoderm by assessing the LacZ expression in a single drop of tail blood obtained at weaning. [16172710]
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).
Temperature: 37.0°C
Growth conditions: Use a feeder layer, LIF and frequent subcultures to prolong the undifferentiated state
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
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 irradiated or Mitomycin C-treated mouse embryonic fibroblasts (MEFs) e.g. ATCC SCRC-1040.1 or SCRC-1040.2a as a feeder layer at approximately 5.0 to 6.0 X 10(6) cells/T75 at least one day before plating the cells (see product sheet for mitotically arrested MEFs for protocol). One hour before thawing the vial of ES cells, perform a 100% medium change using 10 ml of complete ES-DMEM (see below 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 ES cells.
  3. Wash with PBS (Ca+2/Mg+2-free, ATCC SCRR-2201).
  4. Add 3.0 ml of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC 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 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.
  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 ES 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 2 to 3 days
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:7 is recommended
Medium Renewal: Every day.
Preservation: Storage temperature: liquid nitrogen vapor phase
Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO
References: 16172708: Hughes, DE et al. Genetic variation in C57BL/6 ES cell line. Mamm. Genome 18: 549-558, 2007. PubMed: 17828574
16172709: Ware, BC, et al. Utiltiy of a C57BL/6 ES line versus 129 ES lines for targeted mutations in mice. Transgenic Res. 12: 743-746, 2003. PubMed: 14713204
16172710: Graubert A T, et al. Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene. Nucleic Acids Research. 26 (12) :2849-2858, 1998. PubMed: 9611227
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