Macrophage chemotaxis assay

Summary

Macrophage migration is the primary condition for its immune activity at the site of infection and injury, and macrophage chemotaxis reflects its migration ability, which is one of the main indicators for evaluating macrophage immune function. Therefore, for the study of macrophage immune function, it is necessary to establish an effective in vitro chemotaxis assay for the observation of macrophage chemotaxis under different stress states.

Principle

The basic principle of the macrophage chemotaxis assay is that the Transwell is designed according to the ability of the target cells to chemotactically and actively migrate across a certain pore size of the filter membrane.

The membrane separates the chamber into two parts: target cells are on the top and chemokines are on the bottom. The chemokines form a gradient through the membrane, and the cells pass through the membrane pores along the gradient and adhere to the bottom of the membrane, and the chemotactic ability of the chemotactic subcells can be measured by staining and counting the number of cells on the bottom surface of the membrane.

Operation method

Macrophage chemotaxis assay

Principle

The basic principle of the macrophage chemotaxis assay is that the Transwell is designed according to the ability of the target cells to chemotactically and actively migrate across a certain pore size of the filter membrane. The membrane separates the chamber into two parts: target cells are on the top and chemokines are on the bottom. The chemokines form a gradient through the membrane, and the cells pass through the membrane pores along the gradient and adhere to the bottom of the membrane, and the chemotactic ability of the chemotactic subcells can be measured by staining and counting the number of cells on the bottom surface of the membrane.

Materials and Instruments

Reagents:
Chemokines (yeast polysaccharide-activated serum)
Giemsa stain, PBS, methanol
Instruments:
Transwell chemotaxis chamber

Move

The basic procedure of macrophage chemotaxis function assay can be divided into the following steps:

(1) Isolate mouse peritoneal macrophages and adjust them to 2x108/L to 3x108/L with PBS (pH 7.2).

(2) 25 μl of chemokine solution was added to the lower chamber of the chemo-compartment. Cover the hole with the smooth side of the filter membrane with a small corner cut off from the upper left side facing the chemokine.

(3) Cover the upper chamber portion of the chemotaxis chamber and clamp it with a retainer.

(4) Add 50 μl of cell suspension to the upper chamber. Be careful not to generate air bubbles during the operation, so that air bubbles do not hinder the movement of cells.

(5) After each well was injected with monocyte suspension, cover all wells with a 25 mmx80 mm slide. The wells were left at 37 ℃ in a 5% CO2 incubator for 90 minutes to induce cell chemotaxis.

(6) Remove the porous chemotaxis chambers from the incubator; loosen the fixator, place the chambers upside down on a pre-prepared paper pad, and remove the filter membrane.

(7) Secure one side of the filter membrane with large tongs by cutting off a corner beforehand, and clip the other side with small tongs.

(8) Immerse the side of the filter membrane facing the mononuclear cells (i.e., the non-glossy side facing downward) into saline, and gently pull up the filter membrane on one side of the large tongs from the liquid (at an angle of 30° to the container), so as to elute the immobile cells from the filter membrane.

(9) The glossy side of the filter membrane was placed on a slide (40 mmx80 mm) previously coated with egg white glycerol (glycerol mixed with egg white in equal amounts), and the membrane was dried with cold air, fixed with methanol, and subjected to Giemsa or Rieck's staining.

Caveat

(1) It is necessary to add bovine serum albumin or calf serum to the buffer, otherwise it is not effective.(2) If polycarbonate filter membrane is used, attention should be paid to its front and back sides, and the shiny side must be used as the attractant side.(3) The porous chemotaxis chamber device cannot be immersed in organic solvents or detergents, otherwise it will cause dissolution of acrylic resin or deposition of detergents in the pores.Neither can they be put into the oven to bake dry, but can only be rinsed clean with a large amount of running water, then rinsed with double-distilled water and dried naturally.(4) The number of macrophages and the incubation time change with the pore size of the filter membrane, and the optimal conditions should be pre-selected.

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