PluriStem 129/S6 Murine ES cel

PluriStem 129/S6 Murine ES cells

Chemicon (Millipore)

The PluriStem™ 129S6 Murine ES cell line is derived from the widely used 129/S6/SvEv strain of mice (at 3.5 days p.c.) and is intended for injection into black or albino host blastocysts. This cell line is characterized by the production of highly chimeric, germline-competent mice. Cells are supplied at passage 10.

Complete disease testing and karyotype data is available under Information Library tool bar, Tech Notes at SCR012 product specific page at http://www.chemicon.com/Product/ProductDataSheet.asp?ProductItem=SCR012.

STRAIN: 129/S6/SvEv

GENDER: Male

KARYOTYPE: 40XY

Stem Cell Culture

SUBCULTURING:
It is recommended that PluriStem™ 129S6 ES cells be cultured on a monolayer of mitotically inactivated primary mouse embryonic fibroblast cells (MEFs) in the presence of 1000 units / ml of ESGRO® Supplement (mLIF) (ESG1106, ESG1107). PluriStem 129S6 ES cells grow as tightly clustered colonies and should not be allowed to grow to confluence. The colonies should remain small with tight, phase bright borders. Optimal results are achieved when the cells are maintained as small colonies at high density, fed daily and passaged 1:3 to 1:5 every other day.

CULTURE NOTES::
· Incubator settings: 7.5% CO2 in humidified air, 37oC
· Replace the media or passage daily
· Trypsinize gently using 0.05% Trypsin/EDTA

MICROINJECTION NOTES:
· Best results are achieved by injecting 5-7 ES cells per C57BL/6 host blastocyst


Day 0

Prepare the following culture dishes containing a monolayer of mitotically inactivated MEF feeder cells:
· 1 x 25 cm2 flask
· 1 x 75 cm2 flask
· 3 x 10 cm dishes


Day 1

Thaw one vial of PluriStem™ 129S6 ES cells directly into a 25 cm2 flask containing a confluent layer of inactivated MEFs and 5 mls of freshly prepared PluriStem™ 129S6 ES cell media.

· Replace the MEF cell medium with 5.5 mls of PluriStem™ 129S6 medium (see below), and allow it to equilibrate in a 37oC incubator 1 hour before thawing the ES cells.

· Thaw one vial of PluriStem™ 129S6 ES cells by gently shaking the tube in a 37°C water bath. When the contents of the tube have thawed, spray the vial with ethanol , dry outside of vial, and aseptically transfer the contents of the vial to the flask.

· Place the flask in a 37°C incubator overnight.


Day 2

Passage the PluriStem™ 129S6 ES cells to a 75 cm2 flask, containing a confluent layer of inactivated MEFs and 15 mls of pre-warmed PluriStem™ 129S6 ES cell medium.

· Examine the ES cells under the microscope. Many small phase bright ES cell colonies should be visible.

· Replace the MEF cell medium in the prepared 75 cm2 flask with 15 mls of Pluristem 129S6 ES cell medium, and allow the medium to equilibrate in a 37°C incubator for 1 hour before passaging the ES cells.

· Aspirate the medium from the 25 cm2 flask containing the PluriStem™ 129S6 ES cells and rinse with 3 mls of PBS.

· Aspirate the PBS and add 1.5 mls of 0.5% Trypsin/EDTA, place the flask in a incubator for 5 minutes or until the ES cells are dissociated.

· Add 5 mls of PluriStem™ 129S6 ES medium and gently titurate the contents of the flask.

· Transfer the cell suspension to the prepared 75 cm2 flask.

· Place the flask in the 37°C incubator overnight.


Day 3

Electroporation.

· Gently trypsinze the PluriStem™ 129S6 ES cells, as described above, and follow your preferred electroporation protocol.

· Plate the electroporated cells on the prepared 10 cm dishes and begin selection 24 hours following electroporation.


PLURISTEM 129S6 ES CELL MEDIUM MEDIA PREPARATION (250 mL):

DMEM, High Glucose, 200 mL (Millipore cat. no. SLM-020-A or -B)

FCS-ES Cell qualified, 40 mL (Millipore cat. no. ES-009-B or -C)

L. glutamine 200mM, 2.5 mL (Millipore cat. no. TMS-002-C)

Non-Essential AA, 2.5 mL (Millipore cat. no. TMS-001-C)

Pen/Strep, 2.5 mL (Millipore cat. no. TMS-AB2-C)

HEPES (1M), 2.5 mL (Millipore cat. no. TMS-003-C)

MTG, 3.4 μL (Sigma cat. no. M-6145)

ESGRO® (mLIF), 25 μL of ESG1107 or 250 μL of ESG1106

Mix all ingredients in the top of a 250 mL filter (0.22μm PES unit such as Millipore cat. no. SCGPU02RE), and filter sterilize. Store at 4°C. Discard unused media 7-10 days after preparation.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Mouse Embryonic Stem Cells

Cells/Isolation

Embryonic Stem Cells

Cells are supplied frozen in basal medium supplemented with 10% FCS and 10% DMSO

Place vials in liquid Nitrogen immediately upon receipt until it is convenient to proceed to subculture.

2.5 x 10⁶ cells per vial