Human Prostate Cancer-Associated Fibroblasts (CAF)

Catalog Number: HPCAF01
Product Format: Frozen Vial
Cell Number: 1,000,000 cells/vial
Suggested Medium: CAF Growth Medium (CAF01)

General Information

Human Prostate CAF is isolated from human prostate tumor tissue. Cells are grown in T75 tissue culture flasks pre-coated with AlphaBioCoat solution for 30 minutes and incubated in CAF Growth Medium for 3–7 days. Cultures are then expanded. Prior to shipping, cells at passage 1 are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1,000,000 cells per ml.

Product Testing

Human Prostate CAF is tested for negative expression of von Willebrand Factor Expression/Factor VIII, cytokeratin 18, and alpha-smooth muscle actin.
Cells are negative for bacteria, yeast, fungi, and mycoplasma.
Cells express CAF markers FAP, PDGFR, Vimentin, PDPN, and CD70.
Cells can be expanded for 3–5 passages at a split ratio of 1:2 or 1:3.

Laboratory Applications

Human Prostate CAF can be used for the assay of cell-cell interaction, adhesion, PCR, Western blot, immunoprecipitation, immunofluorescent flow cytometry, or for generating cell derivatives for desired research applications.

Shipping

Frozen Vials on Dry Ice

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If continued storage is required, store in the liquid nitrogen vapor phase and not at -70°C, as storage at -70°C will result in loss of viability.

Thaw the vial by gentle agitation in a 37°C water bath. To reduce contamination risk, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).

Post-Thaw Handling Procedure

1. Remove the vial from the water bath once thawed and decontaminate with 70% ethanol. Perform all subsequent steps under strict aseptic conditions.

2. Remove the cryoprotective agent immediately. Centrifuge the cell suspension at 125 × g for 5 to 10 minutes. Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium.

3. Add 6.0 to 8.0 mL of AlphaBioCoat to the T-Flask for 15 minutes. Aspirate the solution, rinse with 8 mL of 1× PBS, and discard the rinse. Transfer cells to the flask.

4. To avoid excessive alkalinity during recovery, pre-incubate the culture vessel containing the growth medium at 37°C for 15 minutes to allow the medium to reach a pH of 7.0 to 7.6.

5. Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended.

Subculturing Procedure

Volumes are for a 75 cm² flask; adjust accordingly for other culture vessels. T-75 flasks are recommended.

Note: Do not hit or shake the flask to detach cells. If needed, place at 37°C to aid in dispersal.

1. Remove and discard the culture medium. Briefly rinse the cell layer with 1× PBS to remove all serum traces.

2. Add 2.0 to 3.0 mL of Cell Detachment Solution. Observe cells under an inverted microscope until the layer is dispersed (typically within 5 to 15 minutes).

3. Add 6.0 to 8.0 mL of AlphaBioCoat to the T-Flask for 15 minutes. Aspirate, rinse with 8 mL of 1× PBS, and discard.

4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gentle pipetting.

5. Dispense appropriate aliquots into new culture vessels.

6. Incubate cultures at 37°C.

• Subcultivation Ratio: 1:2 to 1:3

• Medium Renewal: Every 3 to 4 days

Reagents for Cryopreservation

Complete growth medium supplemented with 5% (v/v) DMSO
Catalog #: CGM5