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Proliferation Bioassay NR6R 3T3 Cells
First Published in R&D Systems 2003 Catalog

Introduction

R&D Systems uses NR6R-3T3 cells in proliferation bioassays to measure the activity of the following cytokines and neutralizing antibodies:

Human Cytokines

  • ß-ECGF
  • FGF acidic
  • FGF basic
  • FGF-4
  • FGF-5
  • FGF-6
  • FGF-8b
  • FGF-8c
  • FGF-17
  • FGF-18
  • PDGF
  • PDGF-AA
  • PDGF-BB
  • PDGF-AB

Bovine Cytokines

  • FGF acidic
  • FGF basic
  • PDGF
  • PDGF-BB

NR6R-3T3 Cell Bioassay Materials


Cell Growth and Preparation
  • NR6R-3T3 cells, a mouse fibroblast cell line (Dr. Angie Rizzino1)
  • 1X Trypsin Solution: Dilute 10X Trypsin EDTA (Irvine Catalog # 9342) 1:10 in PBS
  • Growth Medium: DMEM high Glucose (Irvine Catalog # 9024) 10% Fetal Bovine Serum (Biocell Catalog # 6201)
    2 mM L-glutamine
    100 U/mL Penicillin
    100 µg/mL Streptomycin
NR6R-3T3 Cell Bioassay
  • NR6R-3T3 cells
  • DMEM high Glucose (Irvine Catalog # 9024)
  • Fetal Bovine Serum (Biocel Catalog # 6201)
  • Defibrinated bovine plasma (Irvine Catalog # 1067)
  • PBS (GIBCO Catalog # 14040-133)
  • PBS containing 1 mg/mL BSA (Bayen/Pentex Catalog # 18-068-3)
  • Dialysis tubing (Baxter Catalog # D1614-13, 12,000-14,000 MW cut off)
  • Sodium Heparin (Sigma Catalog # H3149)
  • 3H-thymidine (NEN Catalog # NET027E)
  • Plasma derived bovine serum (PDS) - dialyzed to remove growth factors:
    1. Thaw approximately 100 mL of defibrinated bovine plasma.
    2. Prepare dialysis tubing by cutting it to the appropriate length and softening it in deionized water.
    3. Fill dialysis tubing with defibrinated bovine plasma and seal.
    4. Place dialysis tubing into DMEM. Stir gently on a stir plate for approximately 6 hours at 4 °C. Change the medium by replacing with fresh medium. Dialyze overnight at 4 °C.
    5. Collect dialyzed PDS after 24 hours and filter 2 times through a 0.2 µm filter.
    6. Store extra PDS at -20 to -80 °C.
  • Sodium Heparin Stock
    1. Dissolve sodium heparin in PBS.
    2. Add the sodium heparin solution to DMEM containing 2% PDS (see specific cytokine insert for required final concentration).
  • Growth Medium: DMEM high Glucose 10% Fetal Bovine Serum
    2 mM L-glutamine
    100 U/mL Penicillin
    100 µg/mL Streptomycin
  • Assay Medium: DMEM high Glucose 2% PDS
    2 mM L-glutamine
    100 U/mL Penicillin
    100 µg/mL Streptomycin
Figure 1. Human FGF basic stimulates the 3H-thymidine incorporation by NR6R-3T3 fibroblasts in a dose-dependent manner.1 The ED50 for this effect is typically 0.1 - 0.25 ng/mL.
Figure 2. To measure the ability of the antibody to neutralize the bioactivity of rhFGF basic on NR6R-3T3 fibroblasts, human FGF basic was incubated with various concentrations of the antibody for 1 hour at 37 °C in a 96-well microtiter plate. Following this preincubation period, the antigen-antibody mixture was added to quiescent confluent cultures of NR6R-3T3 cells in DMEM with 2% bovine plasma-derived serum. The assay mixture in a total volume of 100 µL, containing antibody at the concentrations indicated and rhFGF basic at 0.5 ng/mL, was incubated at 37 °C for 20 hours in a humidified CO2 incubator. 3H-thymidine was added during the final 2 hours of incubation. The cells were subsequently detached and harvested onto glass fiber filters and the 3H-thymidine incorporated into DNA was determined. The ND50 of the antibody is approximately 0.5 - 1.0 µg/mL.

Procedure

Cell Growth and Preparation
As with all materials of human source, gloves and lab coats should be worn. All materials contaminated by these cells should be either decontaminated or disposed of in biohazard containers to be autoclaved. All procedures are carried out under sterile conditions.

Doubling Time: Approximately 17 hours Appearance: Rectangular shaped, attached cells

  1. Seed 4 x 105 cells into a 75 cm2 flask containing 20 mL Growth Medium.
  2. Feed the cells every 3 days by replacing the medium with fresh Growth Medium.
  3. Split the cells every week and reseed in fresh Growth Medium. The cells are ready to use in the bioassay when they reach confluency (3 x 106 cells/75 cm2 flask).

NR6R-3T3 Cell Bioassay

  1. Add the cells to each well, leaving the last column empty.
    • Harvest the confluent NR6R-3T3 cells by trypsinization with 1X Trypsin solution.
    • Wash the cells twice with 10 mL Growth Medium.
    • Resuspend the cells in Growth Medium at 0.5 x 105 cells/mL.
    • Add 100 µL of cells to each well, leaving the last column empty.
    • Incubate for 2 days (cells should be 90-100% confluent).
    • Shake out the Growth Medium.
    • Gently add 100 µL Assay Medium to each well.
    • Incubate for 24 hours (cells are now in a resting phase and ready to bioassay).
  2. Reconstitute the cytokine (standard) as directed on each specific cytokine insert. If using rhFGF-5, reconstitute samples in PBS containing 1 mg/mL BSA and 0.1 µg/mL sodium heparin. For assays using rhFGF acidic, rhb-ECGF, rhFGF-5 and rhFGF-6, FGF-8b, FGF-8c, FGF-17, and FGF-18 add 10 µL of Sodium Heparin Stock to each well so that the final concentration of sodium heparin in the assay mixture is as follows:
    • rhFGF acidic, rhb-ECGF, and rhFGF-17 - 10 µg/mL sodium heparin
    • rhFGF-5, rhFGF-8b, rhFGF-8c, and rhFGF-18 - 0.1 µg/mL sodium heparin
    • rhFGF-6 - 1.0 µg/mL sodium heparin
  3. Dilute the standards and samples to their working concentration with Assay Medium.
  4. Add the standards and samples to the plate.
    • Add 50 µL to the first well and serial dilute.
    • Leave the last well with Assay Medium only, as a blank control.
Refer to Figure 2 legend for the neutralizing antibody assay conditions for anti-FGF basic antibody (Catalog # AB-233-NA).
  1. Incubate for 16 - 18 hours at 37 °C with 5% CO2 in a humidified chamber.
  2. Pulse the cells with 0.25 µCi 3H-thymidine/well. Incubate for 2 hours.
    • Prepare 25 µCi/mL 3H-thymidine working stock in Assay Medium.
    • Add 10 µL of 25 µCi/mL 3H-thymidine working stock to each well.
  3. Harvest the cells and count.
    • Wash the cells with 100 µL PBS/well.
    • Add 100 µL 1X Trypsin Solution/well to detach the cells.
    • Harvest on glass fiber filters using a cell harvester.
    • While harvesting, incubate the plate in water for 2 minutes to lyse the cells.
    • Wash the plate thoroughly to ensure all the attached cells have been removed.
    • Count the 3H-thymidine incorporated into the DNA.
    • Plot the dose response using a 4-parameter fit equation.

Troubleshooting

  1. Timing is critical for this assay. Longer or shorter incubation times will shift the ED50.
  2. If the cell doubling time changes, it will change the assay results and ED50.

References

  1. Rizzino, A. et al. (1988) Cancer Research 48:4266-4271.