hTERT Human Lung Cancer-Associated Fibroblasts (CAF) – Squamous Cell Carcinoma

Catalog Number: HT14
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium

General Information

Puromycin-resistant hTERT fibroblasts were selected from HLCCAF following lentiviral transduction of hTERT.
These Human Lung CAFs, derived from squamous cell carcinoma tissue, were cultured in T75 tissue culture flasks pre-coated with AlphaBioCoat and incubated in CAF Growth Medium for 3–7 days. At passage 1, cells were cryopreserved and stored 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

• Mycoplasma, bacteria, yeast, fungi: Not detected

• CAF Marker Expression: FAP, PDGFR, Vimentin, PDPN, CD70

• Expansion Capacity: 3–5 passages at a 1:2 or 1:3 split ratio

Laboratory Applications

• Tumor microenvironment modeling

• Cell-cell interaction assays

• Western blotting, PCR, immunoprecipitation

• Flow cytometry and immunofluorescence

• Custom CAF-based lung cancer models

Shipping

Frozen vials shipped on dry ice

Post-Thaw Recovery Protocol

1. Thawing & Aseptic Handling

   • Thaw the cryovial by gently agitating in a 37°C water bath (~2 minutes).

   • Do not submerge the cap.

   • Decontaminate the vial by spraying or dipping in 70% ethanol.

   • Proceed under strict aseptic conditions.

2. Cell Recovery & Seeding

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

   • Discard the supernatant and resuspend in fresh CAF Growth Medium.

   • Coat a T-flask with 6–8 mL AlphaBioCoat (15 minutes), rinse with 8 mL 1× PBS, discard.

   • Plate cells in the prepared flask.

3. Incubation Conditions

   • Incubate at 37°C in a humidified 5% CO₂ incubator.

   • Pre-equilibrate the growth medium in the incubator for 15 minutes to ensure a pH of 7.0–7.6.

Subculturing Instructions

Volumes below are for 75 cm² flasks. Adjust for other formats accordingly.

1. Remove spent medium.

2. Rinse with 1× PBS to eliminate residual serum inhibitors.

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

   • Do not agitate the flask to avoid clumping.

   • Warm flask or extend incubation if detachment is incomplete.

4. Coat a new flask with AlphaBioCoat (6–8 mL for 15 minutes), rinse with 8 mL PBS, discard.

5. Resuspend cells in 6.0–8.0 mL of complete growth medium.

6. Plate appropriate aliquots into new flasks.

• Subcultivation Ratio: 1:2 to 1:3

• Medium Renewal: Every 3 to 4 days

Cryopreservation Protocol

• Use complete growth medium supplemented with 5% (v/v) DMSO

• Cryopreservation Medium Catalog #: CGM5

• Freeze in isopropanol containers at –80°C overnight, then transfer to liquid nitrogen

Product Usage

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