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…
Divulgaciones
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 |
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An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes. J. Vis. Exp. (Pending Publication), e21897, doi: (2024).