Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Summary

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Abstract

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Introduction

Invasive pulmonary aspergillosis is a great threat to immunocompromised patients as treatment options are limited and only successful upon early diagnosis, which leads to high mortality rates¹. Infectious agents are conidia (spores) of the mold Aspergillus fumigatus that are ubiquitous to most habitats². Conidia are inhaled, pass through the airways and can finally enter the lung alveoli. In immunocompetent humans, these conidia are cleared by innate immune cells such as monocytes or macrophages and neutrophil granulocytes, which take up (phagocytose) and digest the pathogens³. Phagocytos....

Protocol

This protocol includes the use of human buffy coats obtained from the Institute for Transfusion Medicine, Jena University Hospital and fresh venous blood drawn from patients, both after written informed consent of the donors in accordance to ethics committee approval 4357-03/15.

1. Preparation of Aspergillus fumigatus Conidia

1. Grow A. fumigatus on 1.5% malt agar Petri dishes for 5 days at 37 °C without CO₂.
   CAUTION: A. fu.......

Discussion

This protocol presents a fast flow cytometric method to measure interaction of A. fumigatus conidia with a large number of primary human leukocytes that is not possible in common microscopic protocols. Imaging cells with a microscope and manually counting internalized conidia is cumbersome and can realistically be done for a few hundred cells only. Flow cytometry overcomes this problem by measuring thousands of cells within minutes. A hurdle common to both approaches is the distinction of phagocytosed and adhere.......

Materials

Name	Company	Catalog Number	Comments
Adhesive foil	Brand	701367
anti-CD14 V500	BD Biosciences	561391	clone M5E2
anti-CD45 BUV395	BD Biosciences	563792	clone HI30
anti-CD66b PerCP-Cy5.5	BD Biosciences	562254	clone G10F5
anti-FITC APC	ThermoFisher Scientific	17-7691-82	clone NAWESLEE
Cell culture plate, 12-well	Greiner Bio-one	665180
Cell scraper	Bioswisstech	800020
Cell strainer, 30 µm	Miltenyi Biotech	130-098-458	SmartStrainer
Cytometer	BD Biosciences		LSR Fortessa II, lasers: 488 nm (blue), 405 nm (violet), 355 nm (UV) and 640 nm (red)
Detergent	Sigma Aldrich	P1379	Tween 20, 0.01% in PBS
Drigalski spatula	Carl Roth	PC59.1
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	ED3SS-500g	2 mM in PBS
Erythrocyte lysis buffer			0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA
Fetal Calf Serum (FCS)	Biochrom AG	S 0115	10% in RPMI 1640
Fluorescein isothiocyanate (FITC)	Sigma Aldrich	F3651-100MG	0.1 mM in Na2CO3 /PBS solution
Formaldehyd	Carl Roth	PO87.3	Histofix
Malt agar (1.5%)			malt extract (40 g), yeast extract (4 g), agar (15 g), Aqua dest. (1 L), adjust pH to 5.7-6.0, sterilise at 121 °C for 35 minutes
Na2CO3	Carl Roth	8563.1	0.1 M in PBS
Petri dish	Greiner Bio-one	633180
Phosphat Buffered Saline (PBS)	ThermoFisher Scientific	189012-014	without Calcium, without Magnesium
RPMI 1640	ThermoFisher Scientific	61870010	RPMI 1640 Medium, GlutaMAX Supplement
Rotator	Miltenyi Biotech	130-090-753	MACSmix Tube Rotator
Round-bottom tube, 7.5 mL	Corning	REF 352008
Software for data acquisition and analysis	BD Biosciences		FACSDiva 8.0
V-bottom plate, 96 well	Brand	781601	untreated surface