Irradiated Mouse Embryonic Fibroblasts (5 vials) Best Seller
R&D Systems, part of Bio-Techne | Catalog # PSC001
Key Product Details
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Human Pluripotent Stem Cells Cultured on Mouse Embryonic Fibroblasts Express Pluripotent Markers SSEA-4, Oct-3/4, Oct-4A, and E-Cadherin. Human iPS2 stem cells were cultured on Irradiated Mouse Embryonic Fibroblasts (iMEF; Catalog # PSC001). Expression of SSEA-4 was detected using a Mouse Anti-Human/Mouse SSEA-4 Monoclonal Antibody (Catalog # MAB1435) followed by the NorthernLights™ (NL) 493-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog # NL009, green). Oct-3/4 expression was detected using a Goat Anti-Human Oct-3/4 Affinity Purified Polyclonal Antibody (Catalog # AF1759) followed by the NL557-conjugated Donkey Anti-Goat IgG (Catalog # NL001, red). The nuclei were counterstained with DAPI (blue).B.BG01V human embryonic stem cells were cultured on Irradiated Mouse Embryonic Fibroblasts (iMEF; Catalog # PSC001). Expression of Oct-4A was detected using a Mouse Anti-human Oct-4A Monoclonal Antibody (Catalog # MAB17591) followed by the NL493-conjugated Donkey Anti-mouse IgG Secondary Antibody (Catalog # NL009, green). Expression of E-Cadherin was detected using a Goat Anti-Human E-Cadherin Affinity Purified Polyclonal Antibody (Catalog # AF648) followed by the NL 557-conjugated Donkey Anti-Goat IgG (Catalog # NL001, red). Nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips. |
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BG01V Human Embryonic Stem Cells Cultured on Irradiated Mouse Embryonic Fibroblasts Demonstrate a Phenotype that is Consitent with Pluripotency. A. BG01V human embryonic stem cell colonies cultured on Irradiated Mouse Embryonic Fibroblasts (Catalog # PSC001) were analyzed for markers of pluripotency after 3 passages. B. Cells were evaluated for SSEA-4 expression by flow cytometry using a PE-conjugated Mouse Anti-Human SSEA-4 Monoclonal Antibody (Catalog # FAB1435P; filled histogram) or a PE-conjugated Mouse IgG3 Isotype Control Antibody (Catalog # IC007P; open histogram). C.&D. BG01V human embryonic stem cell colonies were incubated with a Goat Anti-Human Oct-3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) or a Goat Anti-Human Nanog Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1997). The cells were stained with the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001). Expression of SSEA-4, Oct-3/4, and Nanog is consistent with a pluripotent phenotype. View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, Irradiated Mouse Embryonic Fibroblasts (iMEFs) can be used as a feeder layer for pluripotent stem cells using the following procedure:
- Thaw iMEFs
- Plate iMEFs onto gelatin-coated plates and culture for 24 hours
- Plate and culture human pluripotent stem cells on iMEF-containing plates
Reagents Supplied in the Irradiated Mouse Embryonic Fibroblasts (Catalog # PSC001):
- 5 vials of irradiated mouse embryonic fibroblasts with 6 x 106 cells/vial
Reagents
- BG01V human embryonic stem cells
- Fetal bovine serum
- Knockout™ serum replacer (Invitrogen)
- Non-Essential Amino Acids (100X)
- L-Glutamine (200 mM)
- Penicillin/Streptomycin (100X)
- beta-mercaptoethanol
- DMEM/F12
- High glucose DMEM
- Recombinant Human FGF basic (Catalog # 233-FB; or Tissue Culture Grade, Catalog # 4114-TC)
- Accutase® (Innovative Cell Technologies)
- 0.1% w/v solution of gelatin in sterile deionized H2O
Materials
- Tissue culture plates (60 mm; the protocol can be adapted for other plate sizes)
- 15 mL centrifuge tubes
- 0.2 µm sterile filter unit
- Pipettes and pipette tips
Equipment
- 37 °C and 5% CO2 incubator
- Centrifuge (low speed clinical or equivalent)
- Hemocytometer
- Microscope
Reagent and Media Preparation
Mouse Embryonic Fibroblast (MEF) Media - MEF media consists of high glucose DMEM, 10% fetal bovine serum, 2 mM L-glutamine, and if desired, add a 1:100 dilution of penicillin/streptomycin (100X) stock. Filter sterilize the media using 0.2 µm sterile filter unit.
Human Embryonic Stem (hES) Media – human embryonic stem cell media consists of DMEM/F12, 15% fetal bovine serum, 5% Knockout serum replacer, 1:100 dilution of non-essential amino acids stock (100X), 1:100 dilution of penicillin/streptomycin (100X) stock, and 0.1 µM beta-mercaptoethanol. Filter sterilize the media using 0.2 µm sterile filter unit. Just prior to use, the media should be supplemented with Recombinant Human FGF basic at 4 ng/mL.
I. Thawing and Plating of iMEF Feeder Cells (plate feeder cells one day prior to stem cell seeding)
Coat the appropriate sized plate(s) with 0.1% sterile Gelatin for 15 minutes.
Aspirate 0.1% Gelatin from plates.
Transfer thawed iMEFs to a 15 mL conical tube that contains 5 mL of pre-warmed Mouse Embryonic Fibroblast (MEF) media.
Centrifuge at 200 x g for 5 minutes.
Remove the supernatant and gently flick the pellet.
Resuspend the iMEF cells in MEF media.
Transfer the cells to the Gelatin-coated plates at a density of approximately 1 x 106 cells/60 mm plate.
Incubate overnight at 37 °C and 5% CO2 incubator before seeding stem cells.
II. Day 2: Thawing and Plating of BG01V Human Embryonic Stem Cells
Transfer thawed BG01V human embryonic stem cells to a 15 mL centrifuge tube that contains at least 5 mL of pre-warmed hES media.
Centrifuge at 200 x g in a clinical centrifuge for 4 minutes.
Remove the supernatant and resuspend the pellet in hES Media supplemented with 4 ng/mL FGF basic.
Remove the MEF media from a plate containing iMEF cells.
Add the BG01V human embryonic stem cell suspension to the plate.
Incubate the cells at 37 °C and 5% CO2.
Replace media daily.
Remove the hES media from BG01V human embryonic stem cells.
Add 1 mL of Accutase solution to each 60 mm plate.
Incubate at room temperature for 5-10 minutes or until cells begin to slough off the plate.
Transfer the cell suspension to a 15 mL centrifuge tube containing >5 mL of pre-warmed hES media.
Centrifuge at 200 x g for 4 minutes.
Remove the supernatant.
Resuspend the pellet in pre-warmed hES media.
Perform a cell count.
Plate the desired number of cells (approximately 0.5-1.0 x 106 cells/60 mm plate) on the iMEF monolayer in hES media supplemented with 4 ng/mL FGF basic.
Replace the media daily.
BG01V cells are licensed from ViaCyte, Inc.