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CellXVivo Human Th1 Cell Differentiation Kit

For the differentiation of Th1 cells from a preparation of naïve CD4+ T cells.
Verification of Th1 Cell Identity using Flow Cytometry. 
(3)
Detection of IFN-gamma, IL-4, and IL-17 in Human Th1 Cells. 
CellXVivo Human Th1 Cell Differentiation Kit
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CDK001

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human CD4+ T cells can be differentiated into Th1 cells using the following procedure:

  • Coat a plate with Mouse Anti-Human CD3 Antibody
  • Isolate human naïve CD4+ T cells from a PBMC preparation
  • Culture naïve CD4+ T cells in Human Th1 Differentiation Media for 5 days
  • Stimulate cells with monensin
  • Analyze culture media and cells for a Th1-specific cytokine production profile
 

 

Reagents Provided

Reagents Supplied in the CellXVivo™ Human Th1 Cell Differentiation Kit (Catalog # CDK001):

  • Mouse Anti-Human CD3 Antibody
  • Human Th1 Reagents
  • Reconstitution Buffers
  • Wash Buffer (20X)

 

Other Supplies Required

Reagents

  • MagCellect™ Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115 or equivalent)
  • Monensin
  • PMA
  • Ficoll-Hypaque™
  • RPMI 1640
  • L-Glutamine
  • Ionomycin
  • Penicillin
  • Streptomycin
  • Fetal Bovine Serum (FBS)
  • beta-Mercaptoethanol

Equipment

  • Tissue culture flasks and/or plates
  • Hemocytometer
  • 37 °C and 5% CO2 humidified incubator
  • Centrifuge
  • Pipettes and pipette tips

 

Procedure Overview

Coat wells of a 24-well plate with Mouse Anti-Human CD3 Antibody.

Coat wells of a 24-well plate

Isolate PBMCs from human blood (e.g., using Ficoll-Hypaque™ density centrifugation).

Isolate PBMCs from human blood

Isolate human naïve CD4+ T cells from PBMCs (e.g., using R&D Systems MagCellect™ Human Naïve CD4+ T Cell Isolation Kit).

Isolate T Cells from PBMCs

Perform a cell count.

Perform a cell count

Suspend 1-2 x 105 naïve CD4+ T cells/mL in media supplemented with Human Th1 Reagent 1 and Human Th1 Reagent 2.
Culture the cells on plates pre-coated with Mouse Anti-Human CD3 Antibody for 5 days.

Refresh the Diferentiation Media every 3-4 days.

Suspend cells in media

Stimulate the cells with mitogens.

Stimulate the cells with mitogens

Verify Th1 cell differentiaiton by analying cytokine expression via flow cytometry.

Verify Th1 cell differentiation

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Product Datasheets

Certificate of Analysis

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