Immortalized Human Glomerular Mesangial Cells (T-HMC)

CAT.NOUNITPRICE
T06951x106 cells / 1.0 ml
$0.00

Specifications


Description

Immortalized Human Glomerular Mesangial Cells (T-HMC) are derived from the kidney of a one month old baby. These cells are immortalized via a double transfection with SV40-T and H-ras oncogenes. T-HMC cells have been shown to express extracellular matrix proteins such as t-PA, PAI1, u-PA, IL-6 and Prorenin. Given that this cell line has retained some of the phenotypic features of human mesangial cells, this particular cell line is very useful to conduct physiological and biological studies of mesangial functions.

SKUT0695
SpeciesHuman (H. sapiens)
Species descriptionHomo sapiens
Tissue/Organ/Organ SystemKidney
Donor GenderDonor Info Not Disclosed
Donor Age1 month old
Donor EthnicityDonor Info Not Disclosed
Growth PropertiesAdherent
Cell MorphologyPolygonal
Immortalization MethodImmortalized by transfection with SV40-T and H-ras
Applications

For Research Use Only

Unit quantity1x106 cells / 1.0 ml
Cell TypeImmortalized Cells
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 II medium, available at abm, Cat. No. TM002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 10%, HEPES to a final concentration of 10 mM, L-glutamine (G275) to a final concentration of 2 mM, and Penicillin/Streptomycin solution (G255) to a final concentration of 1%.

Change media every 2-3 days.
Carbon dioxide (CO2): 5%, Temperature: 37.0°C.
* Do not use heat-inactivated FBS for cell culture unless specified otherwise.

Subculture Protocol

1. Pre-warm the complete growth media specified by the cell line datasheet in a 37°C water bath. 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 the complete media 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 Protocol1. Freeze Medium: 90% FBS and 10% DMSO.
2. Storage Temperature: Liquid nitrogen vapour phase.
QC

1) Immunofluorescence

DepositorGustave Roussy Transfert
FAQs


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 [email protected]
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.
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.
References


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  • Sraer, J., Delarue, F., Hagege, J., Feunteun, J., Pinet, F., Nguyen, G., &, Rondeau, E. "Stable cell lines of T-SV40 immortalized human glomerular mesangial cells" Kidney International 49(1):267-270 (1996). DOI: 10.1038/ki.1996.38.