A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Summary

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Abstract

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Introduction

Neutrophils are the most abundant leukocytes in peripheral blood ¹. During inflammation and infection neutrophils are the first cells to appear at the affected site where they act as the first line of defense ². Neutrophils possess several antimicrobial mechanisms ³ including phagocytosis, production of reactive oxygen species, release of lytic enzymes by degranulation, and production of proinflammatory cytokines ^4,5. Neutrophils are short-lived cells that get rapidly activated through signaling from various cell surface receptors. Although neutrophils have been considered terminal cells due to their short life and because t....

Protocol

1. Isolation of Neutrophils (PMN) from Human Blood

1. Use about 20 ml human blood with heparin (10 U/ml) as anticoagulant. Blood was collected from adult healthy volunteers by venopuncture. All experiments were done under approval of the Bioethics Committee at the Instituto de Investigaciones Biomédicas - UNAM.
2. Put 2 ml of 6% dextran T500 in PBS into a 15 ml conical centrifuge tube and add 10 ml of blood. Mix by inverting the tube two or three times and let it sit for 45 min to allow for erythrocyte sedimentation.
3. In a fresh 15 ml conical centrifuge tube put 5 ml Ficoll-Paque.
4. Take the leukocyte-rich plasma that formed abo....

Results

The purification method described here usually provides unstimulated neutrophils (PMN) with purity greater than 95% (Figure 1A). Isolated PMN can then be stimulated by crosslinking particular receptors with specific monoclonal antibodies. We have stimulated PMN through Fc receptors and integrins (Figure 1B). Once stimulated, PMN are lysed and nuclei are isolated with high yields. Nuclei are then immunolabeled for a particular nuclear factor, such as the nuclear factor κB (NF-κB), wi.......

Discussion

The purification method described here allows the isolation of unstimulated neutrophils (PMN) with purity greater than 95% (assessed by microscopic observation), in a short time. Sometimes neutrophils can be contaminated by erythrocytes if the latter are not lysed completely. This does not usually affect the technique, since erythrocytes and PMN can easily be distinguished as distinct cell populations by flow cytometry. Isolated PMN can then be stimulated by crosslinking particular receptors with specific monoclon.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Heparin	PiSA (Mexico)
Dextran T500	Pharmacosmos A/S (Holbaek, Denmark)	T1-Dextran T500
Ficoll-Paque	Pharmacia	17-0320-01
Sodium chloride	Sigma	S7653
Sodium phosphate monobasic	Sigma	S9638
Sodium phosphate dibasic	Sigma	S9390
Bovine serum albumin (BSA)	Sigma	A2153	Cohn Fraction V
HEPES	Sigma	H3375
Potassium chloride	Sigma	P9541
Magnesium chloride anhydrous	Sigma	M8266
DL-dithiothreitol (DTT)	Sigma	D9163
Trypan Blue (0.4 % solution)	Sigma	T8154
Paraformaldehyde	Sigma	P6148
Triton X-100	Sigma	X100
Fetal bovine serum (FBS)	GIBCO	10437-028
Monoclonal antibody IV.3	Medarex (Annandale, NJ)	025-1	Human-specific anti-FcRII (CD32)
Monoclonal antibody 3G8	Medarex (Annandale, NJ)	028-2	Human-specific anti-FcRIII (CD16)
Monoclonal antibody TS2/16	Dana Farber Cancer Research Institute (Boston, MA)	Donated by Dr. Martin Hemler	Human-specific anti-β1 integrin (CD29)
Monoclonal antibody IB4	University of California, San Francisco	Donated by Dr. Eric J. Brown	Human-specific anti-β2 integrin (CD18)
F(ab')2 goat anti-mouse IgG	Cappel (Aurora, OH)	55468
FITC-conjugated F(ab')2 goat anti-mouse IgG	Cappel (Aurora, OH)	55522
FITC-conjugated F(ab')2 goat anti-rabbit IgG	Cappel (Aurora, OH)	55665
Anti-NF-κB p50	Santa Cruz Biotechnology (Santa Cruz, CA)	sc-114	Rabbit polyclonal antibody
Anti-NF-κB p65	Santa Cruz Biotechnology (Santa Cruz, CA)	sc-109	Rabbit polyclonal antibody
EQUIPMENT
15-ml centrifuge tube	Corning	430791
50-ml centrifuge tube	Corning	430291
Centrifuge, Sorvall Tabletop	Dupont Instruments	RT 6000D
pH-meter	Corning	340
Pipetman pipette P-20	Gilson	F123600
Pipetman pipette P-200	Gilson	F123601
Pipetman pipette P-1000	Gilson	F123602
Hemocytometer	Fisher Scientific	0267110
Microscope	Nikon	Eclipse E600
Inverted microscope	Nikon	TMS
Water Bath Incubator	Fisher Scientific	2IS-M
Microcentrifuge	Eppendorf	5414C
Microcentrifuge	Eppendorf	5418
Flow Cytometer	Becton Dickinson (Franklin Lakes, NJ)	FACScalibur