In-vitro Phagocytosis Assay of Macrophages

Introduction

The term phagocytosis itself describes its mean phage = engulfment; cytosis: cell process. In other words, phagocytosis is the cellular process of engulfing solid particles by the cell membrane to form an internal phagosome, or "food vacuole". The phagosome is usually delivered to the lysosome, an organelle involved in the breakdown of cellular components, which fuses with the phagosome. The contents are subsequently degraded
and either released extracellularly via exocytosis, or released intracellularly to undergo further processing.

Phagocytosis is involved in the acquisition of nutrients for some cells, and in the immune system is a major mechanism used to remove pathogens and cell debris. Bacteria, dead tissue cells, and small mineral particles are all examples of objects that may be phagocytosed. The measurement of phagocytosis activity of immune cells indicates the strength of innate immune system in the host.

Principle

Principle of the phagocytosis assay is simple. Briefly, phagocytic (antigenic; yeast in this case) cells are incubated with the macrophages. The cells were stained with May-Grunwald stain, and attached and engulfed cells were counted with the help of microscope.

Reagents

Preparation of potato dextrose broth

1. Peeled off 200 gm of potatoes and cut into small pieces and keep for boiling in 500 mL water for half an hour.
2. Mince potatoes and filter through a maslin cloth.
3. Add 20 g of dextrose, set the pH to 5.6 and make volume to 1000 mL, and distribut in tubes or flasks.
4. Autoclave the medium at 121ºC and 15 psi for 15 min.
5. Add agar at the rate of 15 g/ L which will be resulted in a solid culture medium potato dextrose agar.
    

DMEM Hams F-12 medium without phenol red

1 tannic acid solution

Working solution of May Grunwald stain

Freshly diluted with Giemsa buffer (1:2)

Yeast cells

1. Use yeast cells for determining phagocytosis. Yeast (Saccharomyces cerevisiae) NCDC40 strain was obtained from NCDC, NDRI.
2. Maintain yeast cells by sub-culturing fortnightly in potato dextrose broth and incubating at 30ºC for 48 h and store under refrigeration between transfers.

Procedure

Preparation of yeast cells

1. Culture yeast cells in potato dextrose broth for 48 h at 30ºC and autoclave at 120ºC for 45 min.
2. Then wash thrice with phosphate buffer saline (PBS) and store at 4ºC until use.
3. Just before use, gently sonicate yeast cells to disrupt clumps and dilute to 10⁸ cells/ mL in DMEM Hams F-12 medium without phenol red.

Phagocytosis assay

1. Collect peritoneal fluid of mice by injecting 5 mL of DMEM Hams F-12 medium (without phenol red) in peritoneal cavity by following a gentle massage of the abdomen.
2. Peritoneal exudate cell suspension (as a source of macrophages) may be used for in vitro phagocytosis assays.
3. Count macrophages in peritoneal fluid using Neubeurâ�#8482;s chamber.
4. Distribute about 1x10⁶ cells/ mL into 35 mm culture grade petri dishes.
5. Incubate in a humidified atmosphere of 5 CO2 in air at 37ºC for 2 h to allow attachment of adherent cells.
6. Remove nonadherent cells by washing 3 times with phosphate buffer saline.
7. After washing, culture cells in DMEM Hams F-12 medium and incubate in a humidified atmosphere of 5 CO2 for 18 h at 37ºC.
8. After incubation, add 100μl of yeast cells (1x10⁸ cells /mL) and incubate for 1 h at 37ºC.
9. Then wash with PBS.
10. Add 1 mL of 1 tannic acid solution and kept for 1 min.
11. After removal of tannic acid solution, again wash with PBS and dry in air.
12. Lastly, stain with May Grunwald stain freshly diluted with Giemsa buffer (1:2) for 2 min.
13. Then wash with PBS and again dry.
14. After drying observe at 1000 x magnification under oil immersion microscope.
15. Calculate percent phagocytosis by counting the number of yeast cells internalized per 100 macrophages.