An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Abstract

Source: Meaney, M. P. et al., Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood. J. Vis. Exp. (2016)

This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Assay Preparation Gather, prepare, and organize the materials specified for the procedure (please refer to Table of Specific Materials/Equipment). Label 12 x 75 mm tubes. Label two tubes for each participant: one tube…

Materials

12 x 75 mm tubes, with cap	VWR	20170-579	You will need 2 tubes per sample ("H" and "F") plus 3 tubes per batch (for assay controls; "F", "H", and "Blood only").
PBS (10X), pH 7.4	Life Technologies	70011-044
Bottle top 0.2 µm cellulose acetate filter (500 ml capacity)	Fisher Scientific	09-741-07
Glucose, powder	Life Technologies	15023-021
Citric acid (anhydrous, cell culture tested, plant cell culture tested)	Sigma-Aldrich	C2404-100G
Sodium citrate tribasic dihydrate	Sigma-Aldrich	S4641-25G
Dihydroethidium (Hydroethidine) (HE)	Life Technologies	D11347
Dimethyl sulfoxide (DMSO) Anhydrous	Life Technologies	D12345
Staphylococcus aureus (Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)	Life Technologies	S2851
Staphylococcus aureus (Wood strain without protein A) BioParticles, unlabeled	Life Technologies	S-2859
Trypan Blue Solution, 0.4%	Life Technologies	15250-061
4.0 mL vacutainer containing 7.2 mg K2EDTA, spray-dried	VWR	BD367861	You will need 1 K2 EDTA blood collection tube per sample.
4.0 mL vacutainer containing 68 USP units Lithium Heparin, spray-coated	VWR	BD367884	You will need 1 Lithium Heparin blood collection tube per sample.
COULTER Ac·T 5diff CP	Beckman Coulter	6605705	i.e., hematology analyzer
COULTER Ac·T 5diff Rinse	Beckman Coulter	8547167
COULTER Ac·T 5diff Fix	Beckman Coulter	8547171
COULTER Ac·T 5diff WBC Lyse	Beckman Coulter	8547170
COULTER Ac·T 5diff Hgb Lyse	Beckman Coulter	8547168
COULTER Ac·T 5diff Cal Calibrator	Beckman Coulter	7547175
COULTER Ac·T 5diff Control Plus	Beckman Coulter	7547198
AcT 5diff Diluent	Beckman Coulter	8547169
200 µL extended-length pipette tips	VWR	37001-526
Insulated ice pan	VWR	89198-980	For ice water bath.
Open metal tube rack	VWR	60916-702
Fetal Bovine Serum	Life Technologies	26140-111
TQ-Prep Workstation	Beckman Coulter	6605429	i.e., automated cell lyse preparation workstation
ImmunoPrep Reagent System	Beckman Coulter	7546999	i.e., RBC lyse/WBC fix reagent system
COULTER CLENZ	Beckman Coulter	8546929