Measuring Intracellular hsp70 in Leukocytes by Flow Cytometry

Linda L. Agnew1

1 Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, Australia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.21
DOI:  10.1002/0471140856.tx0221s49
Online Posting Date:  August, 2011
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Heat shock or stress proteins are constitutively expressed redox‐sensitive proteins, the synthesis of which is induced in almost all organisms exposed to a range of stressors, including heat shock, oxidative stress, free radicals, UV radiation, and heavy metals. This unit details a method, with supporting protocols, for the measurement of their expression in peripheral blood leukocytes by flow cytometry. Curr. Protoc. Toxicol. 49:2.21.1‐2.21.12. © 2011 by John Wiley & Sons, Inc.

Keywords: heat shock; stress proteins; flow cytometry; leukocyte

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Preparation and Staining
  • Alternate Protocol 1: Cell Staining for FACS in 96‐Well Plate Format
  • Basic Protocol 2: Flow Cytometry
  • Support Protocol 1: Collection of Whole Blood
  • Support Protocol 2: Heat Shock
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Cell Preparation and Staining

  • Fluorochrome‐labeled leukocyte extracellular marker antibodies (Becton Dickinson Biosciences), e.g.:
    • anti‐CD3 conjugated to PerCP (cat. no. 347344)
    • anti‐CD3 conjugated to PerCP‐Cy5.5 (cat. no. 340949)
    • anti‐CD4 conjugated to PE‐Cy7 (cat. no. 348789)
    • anti‐CD8 conjugated to APC‐Cy7 (cat. no. 348793)
    • anti‐CD14 conjugated to PE (cat. no. 347497)
    • anti‐CD14 conjugated to APC (cat. no. 340436)
    • anti‐ CD16 conjugated to PE (cat. no. 555407)
    • anti‐CD19 conjugated to PE (cat. no. 555413)
    • anti‐CD36 conjugated to PE (cat. no. 555455)
    • anti‐CD38 conjugated to APC (cat. no. 340439)
    • anti‐CD45RA conjugated to APC (cat. no. 550855)
    • anti‐CD45RO conjugated to PE (cat. no. 555493)
    • anti‐CD56 conjugated to APC (cat. no. 555518)
    • anti‐HLA‐DR conjugated to PerCP (cat. no. 347364)
  • FACS wash buffer (see recipe)
  • Whole blood sample(s), heparinized and heat shocked (see Support Protocols protocol 41 and protocol 52)
  • Whole blood sample, heparinized but not heat shocked (see protocol 4), as control
  • 1× FACS lysing solution (see recipe)
  • 1× FACS permeabilizing solution (see recipe)
  • Fluorochrome‐labeled isotype‐control antibody: an antibody raised in the same species, of the same isotype and with the same fluorochrome as the intracellular hsp antibody, should be used to detect nonspecific binding; if using SPA‐810FI then an anti‐mouse IgG1‐FITC antibody (such as Sigma‐Aldrich F6397) should be used
  • Fluorochrome‐labeled intracellular hsp70 antibody: the antibody should specifically recognize the inducible Hsp70 (HspA1A/HspA1B) (e.g., anti‐hsp70‐FITC, SPA‐810FI, or anti‐hsp70‐ R phycoerythrin (PE) SPA‐810PE; Assay Designs,‐designs/)
  • 1% paraformaldehyde (PFA; see recipe)
  • Polystyrene 12 × 75–mm FACS tubes
  • Centrifuge

Alternate Protocol 1: Cell Staining for FACS in 96‐Well Plate Format

  • 96 well V‐bottom microplates
  • Adhesive plate sealers
  • Centrifuge with microtiter plate adaptors

Basic Protocol 2: Flow Cytometry

  • Animal of interest
  • 10 ml sodium heparin vacutainer blood collection tubes
  • Phlebotomy equipment (e.g., needles and syringes) for venous blood collection

Support Protocol 1: Collection of Whole Blood

  • Heparinized whole blood (from protocol 4)
  • 1.5‐ml microcentrifuge tubes
  • Circulating water bath set at 37°C
  • Circulating water bath set at 42.5°C
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Literature Cited

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