Application protocol

In vitro expansion of mouse CD4+ T cells

Direct ex vivo isolation, cultivation and expansion of CD4+ T cells from mouse spleen
This application protocol describes a reliable workflow for the isolation and cultivation of CD4+ T cells directly from mouse spleen that is fully compatible with downstream applications. Mouse spleens are dissociated into a single-cell suspension using the gentleMACS® Dissociator. Then, CD4+ T cells are magnetically enriched with the CD4+ T cell Isolation Kit, mouse and subsequently activated and expanded with CD3 and CD28 antibody loaded MACSiBeads™ (T cell Activation and Expansion Kit, mouse). Proliferation, phenotype, and expression of activation markers are assessed by flow cytometry at different time points. On day 7 after activation, cells are examined for cytokine expression. Therefore, cells are restimulated with ionomycin and PMA, treated with Brefeldin A and intracellularly stained for cytokines. Additionally, cell culture supernatants are collected 24 h after restimulation and analyzed for cytokines using the MACSPlex Cytokine 10 Kit, mouse. 

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The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.


Buffer (standard wash and dilution buffer)

  • autoMACS® Rinsing Solution (# 130-091- 222)
  • Bovine serum albumin (MACS® BSA Stock Solution; # 130-091-376)

For mouse spleen tissue dissociation

  • Pre-Separation Filters, 30 μm (# 130-041-407)
  • MACSmix™ Tube Rotator (# 130-090-753) 
  • CO2 incubator at 37 °C
  • gentleMACS™  Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes

For isolation of mouse CD4+ T cells

  • CD4+ T Cell Isolation Kit, mouse (# 130-104-454)
  • LS Columns (# 130-042-401)
  • MACS Separator for LS columns (e.g., MidiMACS™ Separator # 130-042-302)
  • MACS Multistand  (#130-042-303)

For flow cytometry analysis of cell proliferation

  • Flow cytometry cell proliferation assay (e.g., CellTrace™ Violet Cell Proliferation Kit from ThermoFisher, # C34557)
  • Phosphate buffered saline (PBS)
  • Fetal bovine serum (FBS) for blocking

For activation and expansion of mouse CD4+ T cells

T cell cultivation

  • TexMACS™ Medium (# 130-097-196)
  • 2-Mercaptoethanol 
  • Mouse IL-2 Research Grade (# 130-098-221)
  • 24-well plate (e.g., Gas-permeable Culture Plate # 150-000-362)
  • 96-well plate (e.g., Gas-permeable Culture Plate # 150-000-364)
  • (Optional) RPMI1640 medium
  • (Optional) 100x L-glutamine stock solution (200 mM)
  • (Optional) 100x penicillin/streptomycin stock solution
  • (Optional) Fetal bovine serum (FBS)

T cell activation and expansion

  •  T cell Activation and Expansion Kit, mouse (#130-093-627)

For flow cytometry analysis of cytokines and surface markers

  • Inside Stain Kit (#130-090-477)
  • Antibodies, mouse:
    CD3e-APC-Vio770 (# 130-105-460)
    CD4-VioGreen™ (# 130-109-413)
    CD62L-PE (#130-102-543)
    CD69-VioBlue® (# 130-103-949)
    CD25-VioBright™ FITC (# 130-108-999)
    CD25-PE-Vio®770 (# 130-108-998)
    CD137-PE (# 130-102-568)
    CD154-APC (# 130-102-454)
    CD134-PE-Vio770 (# 130-109-743)
    CD25-VioBright FITC (# 130-108-999)
    Anti-IL-2-PE (# 130-110-154)
    Anti-TNF-a-FITC (# 130-102-294)
    Anti-IFN-g-PE (# 130-102-388)
  • MACSQuant® Analyzer, MACSQuant Analyzer 10 (# 130-096-343), or other flow cytometers equipped with violet (405 nm), blue (488 nm) and red (635 nm) lasers able to discriminate FITC, PE, and APC fluorescence
    Note: The MACSQuant VYB cannot be used.
  • MACS Chill 96 Rack (# 130-094-459), when using MACSQuant Analyzer or MACSQuant Analyzer 10
  • MACSQuant Calibration Beads (# 130-093-607), when using the MACSQuant Analyzer or MACSQuant Analyzer 10

For removal of MACSiBead™ Particles

  • MACSiMAG™ Separator (#130-092-168)

For restimulation of mouse CD4+ T cells and brefeldin A treatment

  • Ionomycin
  • Phorbol 12-myristate 13-acetate (PMA)
  • Brefeldin A

For flow cytometry analysis of secreted cytokines

  • MACSPlex Cytokine 10 Kit, mouse (# 130-101-740)
  • Polypropylene or polystyrene reagent tubes for serial dilutions of the MACSPlex Cytokine 10 Standard as well as for preparation, dilution, and storage of unknown samples
  • (Optional) Vacuum manifold or centrifuge with adapters to accommodate microtiter plates
  • Orbital shaker for microtiter plates or tubes (frequency 450–1400 rpm)
  • MACSQuant Analyzer, MACS Chill 96 Rack and MACSQuant Calibration Beads (see "For flow cytometry analysis of cytokines and surface markers")
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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.