|Description||Sox17 is a transcription factor that regulates endodermal differentiation of stem cells in the developing human embryo. The endoderm is one of the primary germ layers that appears early in embryonic development. The endoderm gives rise to the lining of the digestive tract, respiratory tract and endocrine organs. Human Sox17 Expressing Embryonic Stem Cells are endoderm progenitors and exhibit markers of the definitive endoderm. Sox17 plays a critical role in general organogenesis of the endoderm during embryonic development and this cell line remains able to undergo hepatic and pancreatic differentiation.|
|Species||Human (H. sapiens)|
|Applications||For Research Use Only|
|Unit quantity||1×106 cells / 1.0 ml|
|Cell Type||Drug Discovery Cell Lines|
|Expression Profile||GATA4, GATA6, HNF4A, FOXA2, CXCR4, CER, GSC, DLX5, ECAD and FOXQ1|
|Propagation Requirements||Use of PriCoatTM T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.
The base medium for this cell line is Prigrow III medium available at?abm, Cat. No.?TM003. To make the complete growth medium, add the following components to the base medium: KnockOut? Serum Replacement (Gibco) to a final concentration of 15% and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.
|Subculture Protocol||1. Pre-warm the Trypsin-EDTA (TM050) to room temperature.
2. Carefully aspirate the culture media from the culture vessel without disturbing the cell monolayer.
3. Add pre-warmed Trypsin-EDTA to the culture vessel. Gently rock the culture vessel to ensure complete coverage of the Trypsin-EDTA over the cells.
4. Observe the cells under a microscope to confirm they are dissociating from each other and are rounding up. Gently tap the culture vessel from several sides to promote cell detachment. Cells that are difficult to detach can be put in 37°C for several minutes to facilitate dispersal.
5. When majority of the cells have detached, add an equal volume of Trypsin Neutralizing Solution into the culture vessel to neutralize the trypsin-EDTA. Gently swirl or pipette the culture suspension to ensure the neutralization is complete.
6. Transfer the culture suspension to a sterile centrifuge tube.
7. Centrifuge the cell suspension at 1500 rpm for 3 minutes. The actual centrifuge duration and speed may vary depending on the cell type.
8. Aspirate the supernatant after checking all cells are pulled down into the pellet. Re-suspend the cell pellet in pre-warmed fresh complete media.
9. Pre-warm new culture vessels to 37°C*. Seed cells at the recommended seeding density.
10. Place the newly seeded culture vessel in a 37°C*, 5% CO2 incubator. Incubate for at least 24 – 48 hours before processing the cells for downstream experiments.
11. Renew the culture media every 2-3 days if the cells have not reached 80% confluency.
* Unless a different temperature is recommended on the data sheet.
|Preservation Protocol||1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO.
2. Storage Temperature: Liquid nitrogen vapour phase.
1. For for-profit organizations and corporations, please contact firstname.lastname@example.org for pricing of this item.
2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at email@example.com.
3. All test parameters provided in the CoA are conducted using abm‘s standardized culture system and procedures. The stated values may vary under the end-user’s culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.
4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination’s temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).
5. All of abm‘s cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.
6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.
7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the “Warranty Period.”
|Depositor||The Hospital For Sick Children|
- Important Considerations for Immortalized Cells
- Immortalized Cell Handling Instructions Upon Arrival
- Subculturing Protocol
- Freezing Protocol
- Hepatocyte Instructions
- Thawing Protocol
I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
How often do I need to change the media?
The media should be changed every 2-3 days.
Why do these cells need bio safety level II?
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in ‘Biosafety in Microbiological and Biomedical Laboratories’ (1999). Your institution’s Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
Do you sell ECM coated T75 flasks?
Yes we can provide a coating service. Please inquire with firstname.lastname@example.org.
Is it necessary that we have to use the recommended T25 ECM-coated flasks for growing the cells? Can we use normal ECM coated 60mm/90mm petri plates for growing the cells?
We strongly recommend using G299 T25 flasks to ensure recovery of these cells before testing other plates.
What can I coat a larger dish to subculture?
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
How long can I store frozen vials for?
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
When are cells plated for live cell shipments?
1 day prior to shipping
Should the cap of the flask be changed before starting the cell culturing step?
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
How is cell density crucial for drug selection?
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don’t need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
My cells are not detaching, what method do you recommend to trypsinize the cells?
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
Why is it important to determine the optimal seeding density?
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
- Séguin, C. A., Draper, J. S., Nagy, A., & Rossant, J. (2008). Establishment of Endoderm Progenitors by SOX Transcription Factor Expression in Human Embryonic Stem Cells. Cell Stem Cell, 3(2), 182–195. https://doi.org/10.1016/j.stem.2008.06.018