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Proliferation Bioassa of TF-1 Cells

Technical Information

Proliferation Bioassa 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 C tokines
C NTF Epo GM-C SF IL-3 IL-4 IL-5 IL-13 LIF b-NGF SC F OSM

Mouse C tokines
IL-5 IL-13 SC F

Rat C tokines
C NTF TF-1 cells will also proliferate in response to human IL-6 and IL-11. TF-1 cells are not responsive to human GC SF, IL-2, IL-7, IL-9 and M-C SF. 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 ATC C (#C RL-2003). The TF-1 cells deposited by Dr. Kitamura with the ATC C 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.

Cell Growth and Preparation

TF-1 human prem eloid cell line Growth Medium: RPMI 1640 (Irvine C atalog #9160) 10% (v/v) Fetal Bovine Serum 2 mM L-glutamine 100 units/mL Penicillin 100 g/mL Streptomycin 2 ng/mL rhGM-C SF (R&D Systems C atalog # 215-GM)

TF-1 Bioassa
TF-1 human er throleukemia cell line

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Proliferation Bioassa of TF-1 Cells

cell line
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TF-1 Bioassa
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 L 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. No e: Refer to figure 2 legend for neutrali ing antibod conditions. 5. Add cells to all wells. Resuspend the cells to 2 x 10 Add 50
5

H-th midine (NEN C atalog # NET027E) Dulbecco's PBS (Irvine C atalog #9240) BSA (Bayer Pentax C atalog # 81068-3) Assa Medium:

assa

RPMI 1640 (Irvine C atalog # 9160) 10% (v/v) Fetal Bovine Serum 2 mM L-glutamine 100 units/mL Penicillin 100 /mL Streptomycin

cells/mL in Assay Medium.

L of cells to each well.

6. Incubate for 48 or 72 hours at 37C with 5% C O 2 in a humidified chamber. 7. Pulse 4 hours with 0.5 Prepare 50.0 Add 10 C i 3 H-thymidine. C i/mL 3 H-thymidine working stock in Assay Medium.

L/well of working stock.

8. Harvest cells and count. C ount 3 H-thymidine labelled DNA. Plot the dose response using a 4-parameter fit equation.

Figure 1. Human GM-C SF stimulates the 3 H-thymidine incorporation by TF-1 cells in a dose-dependent manner (Kitamura, T. et al., (1989) J. C ell Physiol. 140(2):323). The ED 5 0 for this effect is typically 0.02 0.08 ng/mL.

Figure 2. Typical data for anti-hGM-C SF (C atalog # AF-215-NA) is shown. To measure the ability of the antibody to neutralize the bioactivity of rhGM-C SF on human TF-1 cells, rhGM-C SF 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 L, containing antibody at the concentrations indicated, rhGM-C SF at 0.5 ng/mL and cells at 1 x 105 cells/mL, was incubated at 37 C for 48 hours in a humidified C O 2 incubator. 3 H-thymidine was added during the final 4 hours of incubation. The cells were subsequently harvested onto glass fiber filters and the 3 H-thymidine incorporated into DNA was determined. The ND 5 0 of the antibody is approximately 0.08 - 0.16 g/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-C SF 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 characteri ation of a unique human cell line that proliferates dependentl on GM-CSF, IL-3 or er thropoietin. J. C ell Physiol. 140:323-34.

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Proliferation Bioassa of TF-1 Cells

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