Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking

Summary

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the FcγR. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct FcγR cross-linking.

Abstract

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific FcγR stimulation beginning with macrophage generation, priming, staining, FcγR cross-linking, and ending with flow cytometric analysis.

Introduction

Reactive oxygen species (ROS) are reactive molecules or free radicals that are by-products of aerobic respiration (reviewed in ¹). These include the superoxide anion, peroxide, hydrogen peroxide, hydroxyl radical, and hydroxyl ions, among others. Under normal physiologic conditions, ROS are produced mainly by the mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and are rapidly detoxified by various enzymes and proteins such as superoxide dismutase and glutathione. An exaggerated production of ROS or a defect in the ability to remove ROS can result in oxidative stress, whereby reactive oxygen species promote the dama....

Protocol

The protocol for animal handling was approved by the Institutional Animal Care and Use committee (IACUC) of University of Central Florida.

1. Generation of bone marrow derived macrophages (BMDMs)

1. Culture media preparation
   1. Prepare D10F base media: To Dulbecco’s Modified Eagle Medium (DMEM), add 10% heat inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.05 mM β-mercaptoethanol.
      NOTE: Although it is best to use fresh D10F media, D10F base media can be prepared up to two weeks in advance to be used for mu....

Results

Using the protocol outlined within, we present representative data demonstrating flow cytometric detection of ROS production resulting from stimulation of WT C57BL/6J BMDMs through the FcγR. As expected, we observe minimal changes in FL1 or FL2 fluorescence above background levels in unstimulated cells (Figure 3A, compare “stained, unstimulated” vs “unstained, unstimulated” dot plots). We observe a marked increase in FL1 and FL2 fluorescence when cells are stimulated with FcγR cross-linking .......

Discussion

DCFH₂-DA and DHE-based detection of ROS is a widely-used technique^14,15. Ease of use and the adaptability of these ROS probes for kinetic microplate formats, fluorescence microscopy or flow cytometric analysis has contributed to their popularity. However, in our studies of FcγR-mediated macrophage functions, there did not seem to be a standard protocol for performing this assay for flow cytometric analysis of FcγR cross-linked cells. Given th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-BSA IgG1	Innovative Research	IBSA9E2C2
Alexa Fluor 647 Rat IgG2b, κ Isotype Ctrl Antibody	BioLegend	400626
Anti-mouse CD16/32	BioLegend	101302
Anti-mouse F4/80 antibody conjugated to Alexa Fluor 647	BD Biosciences	565853
Anti-mouse F4/80 antibody conjugated to FITC	BioLegend	123108
Anti-mouse/human CD11b antibodyconjugated to Alexa Fluor 647	BioLegend	101218
beta-mercaptoethanol (BME)	Sigma	M3148-100ml
Bovine Serum Albumin (BSA) FractionV	Fisher	BP1600-100
C57BL/6J	Jackson labs	Stock No.000664
CM-H2DCFDA	Molecular Probes	C6827	Can be a substitute for oxidative stress detection reagent in the Enzo kit
Dihydroethidium (DHE)	Molecular Probes	D11347	Can be a substitute for superoxide detection reagent in the Enzo kit
DMEM 1x	Corning	10-013-CV
DMEM no phenol red	Gibco	31053-028
DMF Anhydrous	Acros Organics	61094-1000
Fetal Bovine Serum (FBS)	VWR	97068-085
FITC Rat IgG2a, κ Isotype Ctrl Antibody	BioLegend	400506
HEPES (1M)	Gibco	15630-080
L glutamine	Gibco	25030-081
LADMAC cells	ATCC	CRL-2420
MEM	Corning	10-010-CV
mouse IFN-g	GoldBio	1360-06-100
N-Acetyl-L-cysteine	EMD Milipore	106425	Can be a substitute for ROS inhibitor/scavenger in the Enzo kit
Novocyte flow cytometer with autosampler	Acea	2060R
Pyocyanin (ROS inducer)	Cayman chemical	10009594	Can be a substitute for inducer in the Enzo kit
ROS-ID total ROS/superoxide detection kit	ENZO	ENZ-51010
Sodium pyruvate (100mM)	Gibco	11360-070
Trypsin-EDTA (0.25%)	Gibco	25200-056