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Piezo1 knock-out HEK293T

CRL-3519

The Piezo1 knock-out HEK293T cell line can be used to assess ion channel activity by electrophysiology without any interference from endogenous mechanically activated currents caused by the ion channel Piezo1.
Product category
Human cells
Product type
Cell model
Organism
Homo sapiens, human
Morphology
epithelial
Tissue
kidney
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific applications
These cells allow investigators to assess ion channel activity by electrophysiology without any interference from endogenous mechanically activated currents caused by the ion channel Piezo1.

Characteristics

Cells per vial
Approximately 2.0 to 3.0 x 106
Volume
1.0 mL
Growth properties
Adherent
Comments

These cells allow investigators to assess ion channel activity by electrophysiology without any interference from endogenous mechanically activated currents caused by the ion channel Piezo1.

Note: Do NOT use stacked vessels or roller bottle. Cell detachment may be observed when grown over 80% confluence. The CRL-3519 cell line is very lightly attached to the culture flask and extremely sensitive to media shear effect. Horizontal position should be kept while handling the flask to BSC. Subcultivation must be performed every 2 to 3 days. After washing the flask with PBS, do not remove PBS. The PBS should be transferred to the centrifugation tube to collect the detached cells while rinsing. The tube with PBS washed cells and the tube with neutralized cells can be combined and centrifuged.

Handling information

Complete medium
The base medium for this cell line is Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC 30-2002). To make the complete growth medium, add the following components to the base medium: 10% Fetal Bovine Serum (heat inactivated) (ATCC 30-2020), 2mM L-glutamine (ATCC 30-2214)
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

  1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. Transfer the vial contents to a centrifuge tube containing   9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
  4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
  5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.

         

Subculturing procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.  ;Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Rinse cells with 10 mL PBS . Note: do not remove PBS. The PBS should be transferred to the centrifugation tube to collect the detached cells while rinsing. The tube with PBS washed cells and the tube with neutralized cells can be combined and centrifuged. Add 2.0 to 3.0 mL of 0.25% (w/v) Trypsin- 0.53 mM solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  3. Remove dissociation agent by centrifugation. 150 to 400 x g; 8 to 12 minutes. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  4. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 1.0 x 104 and 4.0 x 104 viable cells/cm2.
  5. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 1.0 X 104 and 3.0 X 104 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: Subcultivation every 2-3 days
Culture maintenance

The flask was seeded with cells (see specific batch information) grown and completely filled with medium at ATCC to prevent loss of cells during shipping.

  1. Upon receipt visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination.  Also check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable).
  2. If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium.  The shipping medium can be saved for reuse.  Incubate the cells at 37°C in a 5% CO in air atmosphere until they are ready to be subcultured.
  3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 x g for 5 to 10 minutes.  Remove shipping medium and save.  Resuspend the pelleted cells in 10 mL of this medium and add to 25 cm2 flask.  Incubate at 37°C in a 5% CO2  in air atmosphere until cells are ready to be subcultured.
Reagents for cryopreservation
DMEM + 10% Heat-Inactivated FBS + 2 mM L-Glutamine + 5% DMSO

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Cytomegalovirus (CMV): Not detected
Epstein-Barr virus (EBV): Not detected
Hepatitis B virus (HBV): Not detected
Human Immunodeficiency virus (HIV): Not detected
Human papillomavirus (HPV): Not detected
STR profiling
Amelogenin: X
CSF1PO: 12,13
D13S317: 12,14
D165539: 9,13
D5S818: 8,9
D7S820: 11
THO1 : 7,9.3
TPOX: 11
vWA: 16,19,20

History

Depositors
Ardem Patapoutian, Dept: Molecular and Cellular Neurobiology, Scripps Research

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Frequently Asked Questions

References

Curated Citations

Lukacs V, et al. Impaired PIEZO1 function in patients with a novel autosomal recessive congenital lymphatic dysplasia. Nat Commun 6:8329, 2015. PubMed: 26387913

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