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

Published: January 31, 2024

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…

Divulgazioni

The authors have nothing to disclose.

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

Tags

Play Video

Citazione di questo articolo
An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes. J. Vis. Exp. (Pending Publication), e21897, doi: (2024).

View Video