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Cytokine Bioassays

Introduction

The biological activity of cytokines is commonly measured by cellular proliferation of primary cell cultures or in vitro adapted cell lines that are dependent and/or responsive to a specific growth factor. Other aspects of biological activity of cytokines include induction of further cytokine secretion, induction of killing, antiviral activity, degranulation, cytotoxicity, chemotaxis, and promotion of colony formation. In vitro assays to measure all of these activities are available. Here, we discuss general protocols for setting up bioassays to measure:

Protocol A: Cytokine-induced proliferation

Protocol B: Cytokine-induced killing

Protocol C: Protection against viral effects

Protocol D: Cytokine-induced cytokine production


Note: Many of the available indicator cell lines are responsive to more than one cytokine (e.g., CTLL2 cells respond to IL-2, IL-4, IL-15) and several cytokines share signaling receptors, so this should be taken into account during experimental design. Neutralizing antibodies for one particular cytokine are useful to demonstrate origin of response and attribute the activity to a particular cytokine. The dependence and responsiveness of cell lines should be carefully ascertained since continuous culture of cells may alter their sensitivity to growth factors. Bacterial endotoxins are effective inducers of some cytokines; hence, their presence in the bioassay cultures should be prevented. In order to obtain statistically significant values, samples should be performed in triplicate wells.

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Protocol A: Cytokine-Induced Proliferation of Indicator Cell Lines

Materials

  • Indicator cell line (see Quick Guide Chart for a given cytokine)
  • Culture Medium (RPMI-1640 supplemented with 10% FBS)
  • 96-well flat-bottom culture plate (Costar Cat. No. 3595)
  • MTT solution (Sigma Cat. No. M5655) 5mg/ml stock in PBS kept at room temperature (protect from light)
  • MTT Lysing Solution 20% SDS/50% DMF

Instruments

  • Pipettes and pipettors
  • Humidified incubator
  • 96-well micro test spectrophotometer

Experiment Duration

Method

  1. Add 100µl of Culture Medium to each well of the 96-well assay plate.
  2. Dilute samples and standard (see Quick Guide Chart for standard range) by 2-fold serial dilution in the assay plate from row 2 to 12. Leave row 1 empty.
  3. Wash indicator cells 3 times with RPMI-1640 and resuspend in Culture Medium at a density of 2-3.5x105 cells/ml (see Quick Guide Chart).
  4. Add 100µl of cell suspension to each well.
  5. Incubate cells for 24-48 hours (see Quick Guide Chart) at 37°C, 5% CO2 in a humidified incubator.
  6. Add 10µl/well of 5mg/ml MTT solution to the plate and incubate for 4 hours.
  7. Add 50µl/well of MTT Lysing Solution to the plate and incubate overnight.
  8. Read plate at 570-650nm.
  9. Graph standard curve and analyze data.

Protocol B: TNF-α-Induced Killing of L929 Cell Line

Materials

  • L929 mouse fibroblast line (ATCC Cat. No. CCL-1)
  • Culture Medium (RPMI supplemented with 10% FBS)
  • Assay Medium (RPMI supplemented with 2% FBS)
  • 96-well flat-bottom culture plate (Costar Cat. No. 3595)
  • Actinomycin D, 500µg/ml stock aliquot kept at minus 80°C (protect from light)
  • MTT solution (Sigma Cat. No. M5655) 5mg/ml stock in PBS kept at room temperature (protect from light)
  • MTT Lysing solution, 20% SDS/50% DMF

Experiment Duration

Method

  1. Prepare L929 cell suspension at a density of 3.5x105/ml in Assay Medium. Add 100µl/well to the 96-well assay plate and incubate overnight at 37°C, 5% CO2 in a humidified incubator.
  2. Dilute samples and standard (see Quick Guide Chart for standard range) by 2-fold serial dilution in the Assay Medium in 100µl/well in another 96-well plate from row 2 to 12. Leave row 1 empty.
  3. Prepare a 4µg/ml working solution of the Actinomycin D by diluting the 500µg/ml stock 125 times in the Assay Medium. Keep Actinomycin D solution protected from light. Add 50µl of this working solution of Actinomycin D to each well.
  4. Transfer 50µl of titrated samples and standard to the corresponding wells on the assay plate.
  5. Incubate plate for 24 hrs at 37°C, 5% CO2 in a humidified incubator.
  6. Add 10µl/well of 5mg/ml MTT solution to each well and incubate for 4 hours.
  7. Add 50µl of MTT Lysing Solution to each well and incubate overnight.
  8. Read plate at 570-650nm. Graph standard curve and analyze data.

Protocol C: IFN-γ Protection from Viral Infection of Cell Lines

Materials

  • L929 mouse fibroblast line (ATCC Cat. No. CCL-1) or A549 human lung carcinoma (ATCC Cat. No. CCL-185)
  • Culture Medium (RPMI supplemented with 10% FBS)
  • Assay Medium (RPMI supplemented with 2% FBS)
  • 96-well flat-bottom culture plate (Costar Cat. No. 3595)
  • EMC virus, 107 pfu/ml aliquots stock kept at minus 80°C
  • MTT solution (Sigma Cat. No. M5655) 5mg/ml stock in PBS kept at room temperature (protect from light)
  • MTT Lysing solution, 20% SDS/50% DMF

Experiment Duration

Method

  1. Prepare L929 or A549 cell suspension at a density of 3.5x105/ml in Assay Medium. Add 100µl/well to the 96-well plate and incubate overnight at 37°C, 5% CO2 in a humidified incubator.
  2. Dilute samples and standard (see Quick Guide Chart for standard range) by 2-fold serial dilution in the assay plate from row 2 to 12. Leave row 1 empty. Incubate the assay plate for 6 hours.
  3. Aspirate supernatant from the wells carefully and add 100µl/well of EMC virus solution at a density of 1-4x104 pfu/ml (see Quick Guide Chart for human IFN-γ: 1/250 dilution of stock of 107 pfu/ml, for mouse IFN-γ, 1/1000 dilution of stock of 107 pfu/ml).
  4. Incubate plate for 40 hours at 37°C, 5% CO2 in a humidified incubator.
  5. Add 10µl of 5mg/ml MTT solution to each well and incubate for 4 hours.
  6. Add 50µl of MTT Lysing Solution to each well and incubate overnight.
  7. Read plate at 570-650nm. Graph standard curve and analyze data.

Protocol D: Cytokine-Induced Cytokine Production

Materials

  • Indicator cell line (see Quick Guide Chart for a given cytokine)
  • Culture Medium (RPMI-1640 supplemented with 10% FBS)
  • 96-well flat-bottom culture plate (Costar Cat. No. 3595)
  • Cytokine ELISA pair or Ready-SET-Go! Reagent Set

Experiment Duration

Method

  1. Add 100µl of Culture Medium to each well of the 96-well assay plate.
  2. Dilute samples and standard (see Quick Guide Chart for standard range) by 2-fold serial dilution in the assay plate from row 2 to 12. Leave row 1 empty.
  3. Wash indicator cells 3 times with RPMI-1640 and resuspend in Culture Medium at a density of 2-3.5x105 cells/ml (see Quick Guide Chart).
  4. Add 100µl of cell suspension to each well.
  5. Incubate cells for 24-48 hours (see Quick Guide Chart) at 37°C, 5% CO2 in a humidified incubator.
  6. Harvest supernatants for determination of cytokine expression by ELISA.
  7. Follow ELISA protocol for target cytokine of interest.

Cytokine Bioassays Quick Guide

The following tables summarize the conditions and reagents needed for human and mouse cytokine bioassays.

Table 1: Mouse Cytokine Bioassays Quick Guide
Mouse Cytokine Bioassays Quick Guide
Cytokine Indicator Cells Cell Density (x105) Feeder Cytokine Assay Medium FBS (%) Incubation Time (hours) Specific Activity U/mg Cytokine Top Conc. (ng/ml) ED50 (pg/ml) Read-Out / Comments
GM-CSF MC/9 (ATCC CRL-8306) 2 mIL-3 (25 U/ml) 10 48 107 10 100 Proliferation
HGF mIMCD-3 0.1 rhEGF (30 ng/ml) 0.05 96 5x104 500 20,000 Proliferation
HMGB1 RAW264.7 3.5   10 24 3x102 100000 3,000,000 TNFa production; with 10ug/ml of Polymycin B
IFNα2 L929 (ATCC CCL-1) 3.5   2 40 107 10 100 EMCV cell protection assay
IFNα4 L929 (ATCC CCL-1) 3.5   2 40 2x107 10 50 EMCV cell protection assay
IFN-γ L929 (ATCC CCL-1) 3.5   2 40 107 10 100 EMCV cell protection assay
IL-1α D10 (ATCC TIB-224) 2 IL-1α (10 pg/ml) + 10% rat ConA supe 10 48 108 1 10 Proliferation
IL-1β D10 (ATCC TIB-224) 2 IL-1α (10 pg/ml) + 10% rat ConA supe 10 48 108 1 10 Proliferation
IL-2 CTLL-2 (ATCC TIB-214) 2 hIL-2 (100 U/ml) 10 24 107 10 80-300 Proliferation
IL-3 MC/9 (ATCC CRL-8306), NFS-60 2 mIL-3 (100 U/ml) 10 48 107 10 80-300 Proliferation
IL-4 CTLL-2 (ATCC TIB-214) 2 hIL-2 (100 U/ml) 10 24 107 10 100 Proliferation
IL-5 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 107 10 100 Proliferation
IL-6 B9 2 hIL-6 (50 pg/ml) 10 48 108 0.1 3.0-12.0 Proliferation; Starve cells for 48 hrs prior
IL-7 hPBMC 2   10 48 2x106 100 200 Proliferation; PHA (5 ug/ml) as costimulator
IL-10 MC/9 (ATCC CRL-8306) 2 mIL-3 (25 U/ml) 10 48 3x106 10 300 Proliferation; mIL-4 (5 pg/ml) as costimulator
IL-12 p70 splenocytes 50   10 24 3x106 10 300 mIFN-γ production
IL-13 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 3x105 100 3,800 Proliferation
IL-15 CTLL-2 (ATCC TIB-214) 3 IL-2 (100 U/ml) 10 48 104 250 100,000 Proliferation
IL-17A NIH/3T3 (ATCC CRL-1658) 2   10 48 3x105 100 3,000 IL-6 production
IL-17A/F NIH/3T3 (ATCC CRL-1658) 2   10 48 105 1000 10,000 IL-6 production
IL-17B PEC 3.5   10 24       TNFa production
IL-17F NIH/3T3 (ATCC CRL-1658) 2   10 48 5x102 100000 2,000,000 IL-6 production
IL-21 B9 2 hIL-6 (50 pg/ml) 10 48 3x106 100 300 Proliferation
IL-22 Colo205 3.5   10 24 107 10 100 IL-10 production
IL-23 splenocytes 50   10 24 107 10 100 mIL-17 production; PMA (5 ng/ml) as costimulator
IL-27 splenocytes 20   10 48 2x104 1000 50,000 Inhibition of IL-2 secretion; immobilized @CD3, soluble @CD28 as costimulators
IL-28 HEPG2 3.5   2 40 107 100 5,000 EMCV cell protection assay
IL-33 D10.G4.1 3.5   10 48 3x107 10 30 Proliferation
SCF MC/9 (ATCC CRL-8306) 2 mGM-CSF (100 ng/ml)   48 - 72 6x104 500 15,000 Proliferation
TNF-α L929 (ATCC CCL-1) 3.5   2 24 2x108 1.0 5.0 L929 cytotoxicity assay; Actinomycin D (2 ug/ml)
VEGF Human umbilical vein endothelial cells 2 rhVEGF (100 U/ml) 10 72 105 1000 8,000 Proliferation
HGF: hepatocyte growth factor
ED50: 50% effective dose of cytokine (1 U/ml observed activity)

Table 2: Human Cytokine Bioassays Quick Guide
Human Cytokine Bioassays Quick Guide
Cytokine Indicator Cells Cell Density (x105) Feeder Cytokine Assay Medium FBS (%) Incubation Time (hours) Specific Activity U/mg Cytokine Top Conc. (ng/ml) ED50 (pg/ml) Read-Out / Comments
GM-CSF TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 107 10 100 Proliferation
HGF 4MBr-5 (ATCC CCL-208) 2 rhEGF (30 ng/ml) 10 96 2x104 1000 50,000 Proliferation
IFN-α1 A549 (ATCC CCL-185) 3.5   2 40 3x106 10 300 A549/EMCV cell protection assay
IFN-α2 A549 (ATCC CCL-185) 3.5   2 40 3x107 10 30 A549/EMCV cell protection assay
IFN-γ A549 (ATCC CCL-185) 3.5   2 40 2.5x107 10 40 A549/EMCV cell protection assay
IL-1α D10 (ATCC TIB-224) 2 IL-1α (10 pg/ml) + 10% rat ConA supe 10 48 5x107 1 20 Proliferation
IL-1β D10 (ATCC TIB-224) 2 IL-1α (10 pg/ml) + 10% rat ConA supe 10 48 107 10 100 Proliferation
IL-2 CTLL-2 (ATCC TIB-214) 2 hIL-2 (100 U/ml) 10 24 107 10 100 Proliferation
IL-3 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 107 10 80-300 Proliferation
IL-4 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 107 10 100 Proliferation
IL-5 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 107 10 100 Proliferation
IL-6 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 107 10 100 Proliferation
IL-7 Human PHA-treated PBMCs (3day) 5   10 48 5x105 100 2,000 Proliferation
IL-8 Human Neutrophils             10-100 ng/ml maximal Chemotaxis
IL-10 MC/9 (ATCC CRL-8306) 2 mIL-3 (100 U/ml) 10 48 106 10 1,000 Proliferation; costimulate with rmIL-4 (5 pg/ml)
IL-12 p70 hPBMC (PHA blast) 2   10 48 107 100 100 Proliferation
IL-13 TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 106 10 1,000 Proliferation
IL-15 CTLL-2 (ATCC TIB-214) 3 IL-2 (100 U/ml) 10 48 107 1 100 Proliferation
IL-17 NHDF 3   10 48 3x105 100 3,000 IL-6 production
IL-21 B9 2 hIL-6 (50 pg/ml) 10 48 105 1000 10,000 Proliferation
IL-22 Colo205 3.5   10 48 107 1 100 IL-10 production
IL-23 Mouse splenocytes 20   10 24 2.5x105 20 4,000 mIL-17 production; costim w/ TPA (5 ng/ml)
IL-27 Mouse splenocytes 20   10 48 2x104 10 50,000 Inhibition of IL-2 secretion
IL-31 U-87               STAT3 activation
IL-33 D10.G4.1 3.5   10 48 3x106 10 300 Proliferation
SCF TF-1 (ATCC CRL-2003) 2 hGM-CSF (100 U/ml) 10 48 5x105 100 2,000 Proliferation
TGF-β HT-2 (CRL-1841) 2 rmIL-2 (100 U/ml) 10 48 107 1 100 Inhibition of IL-4 induced proliferation
TNF-α L929 (ATCC CCL-1) 3.5   2 24 108 10 10 L929 cytotoxicity assay; Actinomycin D (2 ug/ml)
VEGF HUVEC 2 rhVEGF (100 U/ml) 10 72 105 1000 8,000 Proliferation
HGF: hepatocyte growth factor
ED50: 50% effective dose of cytokine (1 U/ml observed activity)

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