Application protocol

In vitro expansion of human Pan T cells  


Isolation, cultivation, and expansion of Pan T cells from human PBMCs  

In this application protocol, pan T cells are isolated directly from human PBMCs by magnetic enrichment using the Pan T Cell Isolation Kit, human and subsequently activated and expanded with the T cell Activation and Expansion Kit, human. T cell purity, proliferation, and expression of activation markers are assessed by flow cytometry at different time points.  

Download Application | PDF 1.2 MB

Protocol

View timeline

This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step. 

Materials

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

1. General

Buffer (standard wash and dilution buffer)

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

2. For isolation of PBMCs

Isolation of PBMCs using Ficoll-Paque™

  • PBS-EDTA: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
    Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
  • Ficoll-Paque (ρ = 1.077 g/mL)
  • 50 mL conical centrifuge tubes  

3. For isolation of human Pan T cells 

Magnetic cell separation

  • Pan T Cell Isolation Kit, human (# 130-096-535)
  • LS Columns (# 130-042-401)
  • MACS Separator for LS Columns (e.g., MidiMACS™ Separator; # 130-042-302)
  • MACS MultiStand (# 130-042-303)  

4. For CFSE labeling for analysis of cell proliferation

  •  5(6)-Carboxyfluorescein diacetate Nsuccinimidyl ester (CFSE; MW 557.46, e.g., from Sigma-Aldrich; # 21888-25MG-F)
  • Dimethyl sulfoxide (DMSO; e.g., CryoMACS® DMSO 10; # 170-076-303)
  • Phosphate-buffered saline (PBS), pH 7.2
  • Human AB serum  

5. For activation and expansion of human Pan T cells

T cell cultivation

  • TexMACS™  Medium (# 130-097-196)
  • Human IL-2 IS, premium grade (# 130-097-744)
  • 24-well plate (e.g. Gas-permeable Culture Plate # 150-000-362)
     ▲ Note: With the Gas-permeable Culture Plate, up to 2.5×107 cells/well/mL can be stimulated as opposed to 1×107 PBMCs/well/mL in standard 24-well plates.
  • (Optional) RPMI 1640 medium
  • (Optional) 100× L-glutamine stock solution (200 mM)
  • (Optional) 100× penicillin/streptomycin stock solution
  • (Optional) 2-Mercaptoethanol
  • (Optional) Fetal bovine serum (FBS)

T cell activation and expansion

  • T Cell Activation/Expansion Kit, human (# 130-091-441)
  • MACSmix™ Tube Rotator (# 130-090-753)

6. For flow cytometric analysis of activation markers, cell proliferation, and expansion rates

  • Antibodies, human:
    CD2-PE (# 130-091-115)
    CD3-FITC (# 130-080-401)
    CD69-APC (# 130-092-159)
    CD25-PE (# 130-091-024)
    CD3-APC-Vio® 770 (# 130-096-610)
  • Propidium Iodide
  • 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.

7. For removal of MACSiBead Particles

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

Matching products: