www.fgks.org   »   [go: up one dir, main page]
Welcome to eBioscience. Tel: 888.999.1371 or 858.642.2058. San Diego, California, USA.
View cart Account Ordering help MSDS
View Cart Account Ordering MSDS
Home BestProtocols® Technical Support About Us Contact Us
Clinical Products Research Products Custom Products Weekly New Products

Cytokine Neutralization, In Vitro

Introduction

Antibodies that block binding of cytokines to their specific receptors and neutralize their effects are critical in studies of cytokine function. The following four protocols describe in vitro bioassays using neutralizing anti-mouse and anti-human cytokine antibodies.

Protocol A: Antibody Neutralization of Cytokine-Induced Proliferation of Indicator Cell Lines

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

Protocol C: Antibody Neutralization of IFN-γ-Protection from Viral Infection of L929 and A549 Cell Lines

Protocol D: Antibody Neutralization of Cytokine-Induced Cytokine Production


In general, the cytokine bioassay protocols are modified to pre-incubate the cytokine of interest with the specific neutralizing antibody prior to addition to the responding cells. This prevents cytokine binding to its receptor on the responding cells, thereby inhibiting the cytokine effect. The chart below summarizes the general optimized experimental conditions for the neutralization assays using indicator cell lines. When working with other cell types, cytokine concentrations, neutralizing antibody concentrations and incubation times may need to be determined by the investigator.

Neutralization Assay Quick Guide Chart

Table 1: Mouse Neutralization Assay Quick Guide Chart
Mouse Neutralization Assay Quick Guide Chart
Specificity Clone Indicator Cytokine Conc. (ng/ml) Ab Top Conc. (ug/ml) ND50 (ng/ml)
GM-CSF MP1-22E9 MC/9 1 10 1250
IFN-γ R4-6A2 L929 1 1 15
IFN-γ XMG1.2 L929 1 1 15
IL-1α ALF-161 D10 4.0 1 31.25
IL-1β B122 D10 4.0 10 125
IL-2 JES6-1A12 CTLL-2 1 1 250
IL-4 11B11 CTLL-2 1 1 125
IL-5 TRFK5 MC/9 1 1 60
IL-6 MP5-20F3 B9 0.01 1 30
IL-10 JES5-2A5 MC/9 1 1 4
IL-12 / IL-23 p40 C17.8 splenocytes 1 1 62
IL-15 AIO.3 CTLL-2 50 10 390
IL-17A eBioMM17F3 NIH/3T3 5.0 1 8
IL-23 G23-8 splenocytes 0.1 10 1250
TNF-α MP6-XT3 L929 1 10 600
TNF-α TN3-19 L929 1 10 150
ND50: 50% neutralizing dose of antibody for given cytokine concentration

Table 2: Human Neutralization Assay Quick Guide
Human Neutralization Assay Quick Guide
Specificity Clone Indicator Cytokine Conc. (ng/ml) Ab Top Conc. (ug/ml) ND50 (ng/ml)
           
IFN-γ NIB42 A549 1 100 5000
IL-1β CRM56 D10 1 10 2500
IL-4 MP4-25D2 TF-1 1 1 30
IL-5 JES1-5A10 TF-1 1 1 15
IL-6 MQ2-13A5 B9 0.05 0.05 0.8
IL-10 JES3-9D7 MC/9 5 1 60
IL-12 / IL-23 p40 C8.6 hPBMC 1 1 60
IL-13 PVM13-1 TF-1 5 1 62
IL-17 eBio64CAP17 NHDF 5 1 15
TNF-α MAb1 L929 1 1 60
ND50: 50% neutralizing dose of antibody for given cytokine concentration

Protocol A: Antibody Neutralization of Cytokine-Induced Proliferation of Indicator Cell Lines

Materials

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

Protocol B: Antibody Neutralization of 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

Instruments

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

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. Add 50µl/well of Assay Medium to each well of another dilution plate.
  3. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 empty (control rows).
  4. Add 50µl/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 empty (cells alone).
  5. Incubate the dilution plate at 37°C, 5% CO2 in a humidified incubator for 2 hours.
  6. Transfer 50µl/well of the Ab/Ag mixed solution to the corresponding well of the assay plate containing cells.
  7. Prepare a 4µg/ml working solution of the Actinomycin D by diluting the 500µg/ml stock 125X in the Assay Medium. Keep Actinomycin D solution protected from light. Add 50µl of this working solution of Actinomycin D to each well.
  8. Incubate plate for 24 hrs at 37°C, 5% CO2 in a humidified incubator.
  9. Add 10µl/well of 5mg/ml MTT solution to each well and incubate for 4 hours.
  10. Add 50µl of MTT Lysing solution to each well and incubate overnight.
  11. Read plate at 570-650nm.
  12. Graph standard curve and analyze data.

Protocol C: Antibody Neutralization of IFN-γ-Protection from Viral Infection of L929 and A549 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

Instruments

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

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 assay plate and incubate over night at 37°C, 5% CO2 in a humidified incubator.
  2. Add 50µl/well of Assay Medium to each well of anther dilution plate.
  3. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 empty (control rows).
  4. Add 50µl/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 empty (cells alone).
  5. Incubate the dilution plate at 37°C, 5% CO2 in a humidified incubator for 2 hours.
  6. Transfer 50µl/well of the Ab/Ag mixed solution to the corresponding well of the assay plate containing cells.
  7. Incubate the assay plate for 6 hours.
  8. 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).
  9. Incubate plate for 40 hours at 37°C, 5% CO2 in a humidified incubator.
  10. Add 10µl of 5mg/ml MTT solution to each well and incubate for 4 hours.
  11. Add 50µl of MTT Lysing solution to each well and incubate overnight.
  12. Read plate at 570-650nm.
  13. Graph standard curve and analyze data.

Protocol D: Antibody Neutralization of 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

Instruments

  • Pipettes and pipettors
  • Humidified incubator
  • 96-well micro test spectrophotometer (ELISA plate reader)
  • ELISA plate washer

Experiment Duration

Method

  1. Add 50µl of Culture Medium to each well of the 96-well assay plate.
  2. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 empty (control rows).
  3. Add 50µl/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 empty (cells alone).
  4. 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).
  5. Add 100µl of cell suspension to each well.
  6. Incubate cells for 24-48 hours (see Quick Guide Chart) at 37°C, 5% CO2 in a humidified incubator.
  7. Harvest supernatants for determination of cytokine expression by ELISA.
  8. Follow ELISA protocol for target cytokine of interest.

Home  |   Index  |   Site Map  |   FAQ  |   Terms & Conditions  |   Distributors  |   Careers  |   Contact Us
Copyright © 2000-2010 eBioscience, Inc.
Unless indicated otherwise, all products are For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for further distribution without written consent. Not all products available in all regions.