Proliferation Bioassay of TF-1 Cells

Contents

• Introduction
• Materials
• Procedure
• Troubleshooting
• Reference

Introduction

TF-1 cells are a factor-dependent human erythroleukemic cell line. TF-1 cells are employed in proliferation bioassays by R&D Systems for the routine quality assurance and quality control of the following cytokines and neutralizing antibodies:

Human Cytokines

• CNTF
• Epo
• GM-CSF
• IL-3
• IL-4
• IL-5
• IL-13
• LIF
• b-NGF
• SCF
• OSM

Mouse Cytokines

• IL-5
• IL-13
• SCF

Rat Cytokines

• CNTF

TF-1 cells will also proliferate in response to human IL-6 and IL-11. TF-1 cells are not responsive to human G-CSF, IL-2, IL-7, IL-9 and M-CSF. The TF-1 cells used by R&D Systems were obtained from Dr. Toshio Kitamura in 1989. Dr. Kitamura has deposited TF-1 cells with ATCC (#CRL-2003). The TF-1 cells deposited by Dr. Kitamura with the ATCC are described as non-responsive to IL-5. If you need to obtain TF-1 cells that will respond to IL-5, please contact R&D Systems Technical Service at 1-800-343-7475.

Procedure

Cell Growth and Preparation

As with all materials of human source, gloves and lab coat 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 30 hours.

Appearance: Single cells, slightly irregular in size and shape. A small percentage will attach to the flask.

1. Seed cells at 5x10⁴ cells/mL or higher in Growth Medium.
2. Split cells every 3-4 days and reseed in fresh Growth Medium.

TF-1 Bioassay

1. Reconstitute standards and samples in PBS + 1.0 mg/mL BSA.
2. Dilute standards and samples to working concentration (6X higher than desired final concentration) with Assay Medium.
3. Add 50 µL of Assay Medium to each well of 96-well plate.
4. Add standards and samples to plate.
   • Leave last well with dilution media only as a blank.
   • Run samples in duplicate.
   Note: Refer to figure 2 legend for neutralizing antibody assay conditions.
5. Add cells to all wells.
   • Resuspend the cells to 2 x 10⁵ cells/mL in Assay Medium.
   • Add 50 µL of cells to each well.
6. Incubate for 48 or 72 hours at 37°C with 5% CO₂ in a humidified chamber.
7. Pulse 4 hours with 0.5 µCi ³H-thymidine.
   • Prepare 50.0 µCi/mL ³H-thymidine working stock in Assay Medium.
   • Add 10 µL/well of working stock.
8. Harvest cells and count.
   • Count ³H-thymidine labelled DNA.
   • Plot the dose response using a 4-parameter fit equation.

for this effect is typically 0.02 - 0.08 ng/mL.

Figure 2. Typical data for anti-hGM-CSF (Catalog # AF215) is shown. To measure the ability of the antibody to neutralize the bioactivity of rhGM-CSF on human TF-1 cells, rhGM-CSF was incubated with various concentrations of the antibody for 1 hour at 37° C in a 96-well plate. Following this preincubation period, TF-1 cells were added. The assay mixture in a total volume of 100 µL, containing antibody at the concentrations indicated, rhGM-CSF at 0.5 ng/mL and cells at 1 x 105 cells/mL, was incubated at 37° C for 48 hours in a humidified CO2 incubator. 3H-thymidine was added during the final 4 hours of incubation. The cells were subsequently harvested onto glass fiber filters and the 3H-thymidine incorporated into DNA was determined. The ND50 of the antibody is approximately 0.08 - 0.16 µg/mL.

Troubleshooting

1. Variations in cell feeding and splitting (growth times) can influence stability of the cell line. Splitting cells at regular intervals and avoiding overgrowth of cells will reduce the possibility of a population of cells becoming GM-CSF growth independent and overtaking the dependent cell population.
2. Keep timing of assay consistent.
3. Always include the correct controls.

Reference

1. Kitamura, T. et al. (1989) Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3 or erythropoietin. J. Cell Physiol. 140:323-34.