Immortalized Human Proximal Tubule Cells (HPCT-03ts)

CAT.NOUNITPRICE
T07485x105 cells / 1.0 ml
$1,065.00

Specifications


Description

Water and electrolyte homeostasis is largely regulated by the human proximal tubule; however this complex system has been hard to study particularly in relation to specific genes and their influence on renal physiology and pathophysiology. Immortalized Human Proximal Tubule Cells (HPCT-03ts) is a potential model to acquire detailed physiological information about cell behaviour in response to hormones, growth factors, nutrients, and other external stimuli.

SKUT0748
SpeciesHuman (H. sapiens)
Tissue/Organ/Organ SystemKidney
Donor GenderFemale
Donor Age75 years old
Growth PropertiesAdherent
Cell Morphology SpecificationEpithelial-cobblestone
Seeding Density150,000 cells/cm2
Population Doubling Time24 - 48 hours
Immortalization MethodImmortalized with retrovirus containing temperature-sensitive SV40 large T antigen
Applications

For Research Use Only

Mammalian Selection MarkerNeomycin
Unit quantity5x105 cells / 1.0 ml
Cell TypeImmortalized Cells
Expression Profile

NBCe1-A, NHE3

Propagation Requirements

Cells are grown on Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 μm (Millipore) that are lightly coated with a 20% dispersion of rat-tail collagen (in 60% ethanol) plus 1 μg/mL laminin.
The base medium for this cell line is Prigrow IV medium available at?abm, Cat. No.?TM004. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 5%, HEPES (Sigma) to a final concentration of 15 mM, sodium selenite (Sigma) to a final concentration of 20 nM, ascorbic acid-2-phosphate (Sigma) to a final concentration of 50 mM, transferrin (Sigma) to a final concentration of 5 μg/mL, Insulin Solution (TM053) to a final concentration of 5 μg/mL, mouse EGF (Sigma) to a final concentration of 10 ng/mL, dexamethasone to a final concentration of 4 μg/mL, T3 (Sigma) to a final concentration of 1 nM, and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.
Change media every 2-3 days by first removing it from the bottom of the filter and then from the top. Add fresh medium first to the top and then to the bottom.
Carbon dioxide (CO2): 5%, Temperature: 33.0°C. To differentiate cells, increase temperature to 38.0°C.
* Do not use heat-inactivated FBS for cell culture unless specified otherwise.

Subculture Protocol

Subculture Protocol (1 filter 0.6 cm2):
1. Aspirate medium, then add Hank’s BSS without calcium or magnesium for 30 seconds to 2 minutes.
2. Replace HBSS with trypsin-EDTA (0.05%) for 15 minutes or less at 37°C.
3. When the cells are separated and rounded, gently pipette to release from filter.
4. Neutralize trypsin with FBS and wash with additional medium to remove any remaining cells.
5. Centrifuge cells at 1,200x g for 3 minutes.
6. Plate in prepared collagen/laminin coated culture inserts.
7. Use minimal apical medium of cells of about 50 to 100 uL/culture insert.

Preservation Protocol1. Freeze Medium: Complete growth medium with 20% FBS in CS10 (Sigma). Follow protocol for CS10.
2. Storage Temperature: Liquid nitrogen vapour phase.
QC

1) Western blot

Disclaimer

1. For for-profit organizations and corporations, please contact [email protected] 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 protected].

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."

DepositorCase Western Reserve University
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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