hTERT Human Lung Cancer-Associated Fibroblasts (CAF) – Adenocarcinoma

Catalog Number: HT15
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium

General Information

Puromycin-resistant hTERT cells were derived from HLCCAF following infection with lentiviruses expressing hTERT.
These Human Lung CAFs were isolated from lung adenocarcinoma tumor tissue and cultured in AlphaBioCoat-coated T75 flasks. Following expansion in CAF Growth Medium (3–7 days), cells at passage 1 were cryopreserved. 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

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

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

Laboratory Applications

• Tumor-stroma interaction studies

• Cell adhesion and migration assays

• PCR, Western blot, immunoprecipitation

• Immunofluorescence and flow cytometry

• Development of CAF-based in vitro models

Shipping

Frozen vials shipped on dry ice

Thawing and Culture Protocol

1. Thawing & Decontamination

   • Thaw vial by gentle agitation in a 37°C water bath for ~2 minutes.

   • Avoid submerging the cap.

   • Disinfect with 70% ethanol.

   • Perform all following steps under sterile conditions.

2. Recovery & Seeding

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

   • Discard the supernatant and resuspend the pellet in fresh CAF medium.

   • Pre-coat a T-flask with 6–8 mL AlphaBioCoat for 15 minutes. Rinse with 8 mL 1× PBS and discard.

   • Transfer the cell suspension to the coated flask.

3. Culture Conditions

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

   • Pre-warm medium in the incubator for 15 minutes to stabilize pH (7.0–7.6).

Subculturing Instructions

Based on a 75 cm² flask. Adjust volumes for other sizes.

1. Discard used medium.

2. Rinse cells with 1× PBS to remove residual serum.

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

   • Do not shake or tap the flask to avoid clumping.

   • Difficult-to-detach cells may require extended incubation.

4. Pre-coat a new T-flask with AlphaBioCoat (6–8 mL, 15 min), rinse with PBS, discard.

5. Add 6.0–8.0 mL of fresh medium to the detached cells and gently aspirate to create a single-cell suspension.

6. Transfer appropriate aliquots to new flasks.

• Subcultivation Ratio: 1:2 to 1:3

• Medium Renewal: Every 3–4 days

Cryopreservation Protocol

• Freeze cells at 1–5 × 10⁶ cells/mL in complete medium supplemented with 5% DMSO

• Use a freezing container (isopropanol-based) to cool gradually at –80°C overnight

• Transfer to liquid nitrogen for long-term storage

• Cryopreservation Medium Catalog #: CGM5

Product Usage

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