Intracellular Cytokine Detection by Flow Cytometry in Surface Marker‐Defined Human Peripheral Blood Mononuclear T Cells

Fredine T. Lauer1, Jesse L. Denson1, Ellen Beswick2, Scott W. Burchiel1

1 Department of Pharmaceutical Sciences, College of Pharmacy, The University of New Mexico, Albuquerque, 2 Department of Molecular Genetics and Microbiology, School of Medicine, The University of New Mexico, Albuquerque
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.19
DOI:  10.1002/cptx.26
Online Posting Date:  August, 2017
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In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi‐color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T‐cells using multi‐color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker‐defined T cells that identify functional subsets of cells. There were no observed differences between fresh and cryopreserved HPBMC in eight phenotypes analyzed (T‐CD3, Th‐CD4, Tmem‐CD45RO, activated T‐CD3/CD25, Treg‐ Foxp3/CD25, Th1‐IFNγ, Th2‐ IL‐4, Th17‐IL‐17A). There was an observed difference in activated T‐ CD3/CD69 in the short term (30‐90 days) cryopreserved samples as compared to the freshly isolated samples, which may have resulted from the variance in controls or small sample size. © 2017 by John Wiley & Sons, Inc.

Keywords: intracellular staining; flow cytometry; immunophenotyping; HPBMC; toxicology

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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Intracellular Markers Using 11‐Color Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Detection of Intracellular Markers Using 11‐Color Flow Cytometry

  • Cells
  • RPMI complete medium (cRPMI; see recipe)
  • Anti‐CD3 Functional Grade Purified (eBioscience, cat. no 16‐0037‐85) (Ab)
  • Dulbecco's phosphate‐buffered saline without calcium or magnesium (DPBS‐)
  • Anti‐CD28 Functional Grade Purified (eBioscience, cat. no. 16‐0289‐85)
  • Brefeldin A Ready Made Solution (BFA; Sigma‐Aldrich, cat. no. B5936)Dimethyl sulfoxide (DMSO)
  • Staining buffer (see recipe)
  • Anti‐mouse Ig, k/Negative Control Compensation Particles Set (BD Biosciences Comp Bead, cat. no. 552843)
  • Brilliant stain buffer (BSB; BD Biosciences, cat. no. 563794)
  • Cell surface marker antibodies (see Table 18.19.1)
  • Ice
  • Fixable viability stain 620 (FVS; BD Biosciences, cat. no. 564996)
  • TF Fix/Perm buffer (see recipe)
  • TF Perm/Wash (see recipe)
  • Intracellular marker antibody (see Table 18.19.1)
  • Anti‐mouse Ig, k/Negative Control Compensation Particles Set (BD CompBeads, cat. no. 552843)
  • Biosafety cabinet
  • Biohazard waste receptacle
  • 35–mm petri dishes
  • 37°C, 5% CO 2 incubator
  • 96‐well, flat‐bottom cell culture plate for CD3/CD28 stimulation of ICS
  • Plastic wrap
  • Sterile 15‐ml conical centrifuge tubes
  • Sterile transfer pipette
  • Centrifuge
  • Multichannel pipette
  • Plate shaker
  • 96‐well polypropylene tubes (cluster tubes; 1.2 ml) in rack with box (Corning, cat. no. 4410)
  • Tips sterile to fit pipettes
  • Refrigerated centrifuge with plate carriers (for centrifuging cluster tubes in the rack/box)
  • Vortex mixer
  • 1.7 ml Microcentrifuge tube or other tube with adequate capacity for mixing antibody cocktail
  • Aluminum foil
  • LSR Fortessa (cell analyzer, BD Biosciences) (Lasers: Blue 488 nm, Yellow/Green 561 nm, Red 640 nm, Violet 405 nm)
  • 12 × 75–mm (5 ml) polystyrene Round bottom tubes (flow tubes; Falcon, cat. no. 352008)
  • Rack/holder for 12 × 75 mm tubes
  • Dual‐position snap caps for 12 × 75–mm tube (Falcon, cat. no. 352032)
  • Vacuum aspirator
Table 8.9.1   MaterialsIntracellular Staining Markers Used in protocol 1Basic Protocol a

Laser Filter Fluorochrome Specificity Clone Cat. no. Amount Ab
Blue 488 nm 530/30 FITC IFNg B27 552887 20 µl
695/40 PerCP‐Cy5.5 IL‐17 A N49‐653 560799 5 µl
Yellow ‐Green 561 nm 582/15 PE IL‐4 8D4‐8 559333 20 µl
610/20 PE‐CF594 FVS620 564996 1 µl
780/60 PE‐Cy7 CD127 HIL‐7R‐M21 560822 5 µl
Red 640 nm 670/14 Alexa Fluor 647 FoxP3 259D/C7 560045 20 µl
730/45 Alexa Fluor 700 CD4 RPA‐T4 557922 5 µl
780/60 APC‐Cy7 CD69 FN50 557756 5 µl
Violet 405 nm 450/50 BV421 CD45RO UCHL1 562641 5 µl
525/50 BV510 CD3 UCHT1 563109 5 µl
605/12 BV605 CD25 2A3 562660 5 µl

 aTable contains information on fluorescent labeled antibodies as well as the amounts used for cell surface and intracellular staining. Flow cytometer laser and filter requirements are also listed. All antibodies used for staining were purchased from BD Biosciences.
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Literature Cited

Literature Cited
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