hTERT Human Kidney Cancer-Associated Fibroblasts (CAF)

Catalog Number: HK01
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium

General Information

Puromycin-resistant hTERT cells are selected from HRMVPCs after lentiviral transduction with hTERT.
These Human Kidney CAFs are isolated from kidney tumor tissue and expanded in T75 flasks coated with AlphaBioCoat. After incubation in CAF Growth Medium for 3–7 days, cells are harvested at passage 1, cryopreserved, and shipped frozen. Each vial contains ≥1,000,000 viable cells.

Product Testing

• Negative for: von Willebrand Factor/Factor VIII, cytokeratin 18, and alpha-smooth muscle actin

• Free from: bacteria, yeast, fungi, and mycoplasma

• Positive for CAF markers: FAP, PDGFR, Vimentin, PDPN, and CD70

• Expandable for 3–5 passages at a split ratio of 1:2 or 1:3

Laboratory Applications

• Cell-cell interaction assays

• Adhesion studies

• PCR, Western blot, and immunoprecipitation

• Immunofluorescence and flow cytometry

• Derivation of CAF-based experimental models

Shipping

Frozen vials on dry ice

Post-Thaw Culture Instructions

1. Thawing & Decontamination

   • Thaw vial by gentle agitation in a 37°C water bath (~2 min).

   • Avoid submerging the cap and O-ring.

   • Spray or dip the vial in 70% ethanol.

   • Perform all steps under strict aseptic conditions.

2. Recovery & Seeding

   • Centrifuge cell suspension at 125 × g for 5–10 minutes.

   • Discard supernatant and resuspend pellet in fresh growth medium.

   • Pre-coat a T-Flask with 6–8 mL AlphaBioCoat for 15 minutes.

   • Rinse with 8 mL 1× PBS and discard.

   • Transfer cells into the prepared flask.

3. Culture Conditions

   • Place culture in a 37°C incubator with 5% CO₂.

   • Pre-warm the culture medium for 15 minutes prior to use to ensure proper pH (7.0–7.6).

Subculturing Protocol

Volumes below are for 75 cm² flasks. Adjust as needed for other sizes.

1. Remove spent medium.

2. Rinse gently with 1× PBS to remove serum inhibitors.

3. Add 2.0–3.0 mL Cell Detachment Solution. Incubate 5–15 minutes at 37°C.

   • Do not shake or tap the flask.

   • For difficult detachment, extend incubation or warm flask.

4. Add 6.0–8.0 mL AlphaBioCoat to a new flask for 15 minutes. Rinse with 8 mL PBS and discard.

5. Add 6.0–8.0 mL fresh growth medium. Aspirate cells by gentle pipetting.

6. Plate into new vessels.

• Subcultivation Ratio: 1:2 to 1:3

• Medium Renewal: Every 3–4 days

Cryopreservation

• Medium: Complete growth medium + 5% DMSO

• Catalog #: CGM5

Product Usage

Cells are offered for Research Use Only. Not for Clinical Use.