Intraperitoneal injection of sodium thioglycolate in mice can stimulate the aggregation of macrophages. Four days later, mice were injected intraperitoneally with sheep erythrocyte suspension, and one hour later, the abdominal phagocytes were dissected and collected, and the phagocytosis of sheep erythrocytes could be observed by staining and microscopic examination. The phagocytosis function of phagocytes can be determined by calculating the phagocytosis percentage or phagocytic index. (Source: Immunology Internship Guide - Department of Immunology, College of Basic Medical Sciences, Peking University)
Operation method
Phagocytic index assay
Principle
Phagocytes have the function of phagocytosis and digestion of foreign substances (bacteria, sheep erythrocytes, chicken erythrocytes, etc.), and play an important role in nonspecific immunity of the organism. Intraperitoneal injection of sodium thioglycolate in mice stimulated the aggregation of macrophages. Four days later, mice were injected intraperitoneally with sheep erythrocyte suspension, and one hour later, the abdominal phagocytosis cells were dissected and collected, and the phagocytosis phenomenon on sheep erythrocytes could be observed by staining and microscopic examination. The phagocytosis function of the phagocytes could be determined by calculating the phagocytosis percentage or phagocytic index.
Materials and Instruments
ICR Mouse Move 1. Preparation for the experiment: 3 ml of 3% sodium thioglycolate was aspirated with a sterile syringe and injected into the peritoneal cavity of mice. Caveat 1. Rub the peritoneal cavity as much as possible to wash down the phagocytes. 2. When aspirating the peritoneal fluid with a pointed pipette, avoid the abdominal organs as much as possible, so as to avoid damaging the blood vessels and causing bleeding, which will affect the experimental results. 3. When staining with Rachel's stain, do not pour off the stain before rinsing, so as to avoid the fine particles in the stain adhering to the slide and affecting the clarity of the specimen. Common Problems For more product details, please visit Aladdin Scientific website.
PBS buffer Sodium thioglycolate Sheep erythrocyte suspension Roche dye Methanol
Anatomical equipment Syringes Tip pipettes Rubber tips Test tubes Slides
2. Four days later, 1 ml of 1% sheep erythrocyte suspension was injected into the peritoneal cavity of the mice.
3. One hour after the injection, the mice were killed by cervical dislocation, and dissected and exposed to expose the abdominal cavity, which was located on the upper part of the abdominal cavity. Use a sharp pipette to inject 5 ml of pre-warmed PBS into the peritoneal cavity, and rub the peritoneal cavity with hands repeatedly for about 1-2 minutes, in order to flush out as many phagocytes as possible from the peritoneal cavity of the mouse.
4. Aspirate the peritoneal cavity fluid with a pointed pipette, and put it into a clean test tube.
5. Blow the peritoneal cavity fluid with a pointed pipette to make it homogeneous (try to avoid air bubbles), and then aspirate a drop of it onto a slide, and then put it onto a coverslip for microscopic examination.
6. Observe the results, or you can also put the peritoneal cavity fluid smears on a flat surface and incubate them for 30 minutes at 37℃. incubate at 37°C for 30 minutes. Remove the smear, rinse 3 times with pre-warmed PBS, then fix with methanol for 1 minute, mix 1 part of Rachel's stain with 2 parts of PBS at pH 6.4, then put a drop of the stain on the smear for 5-10 minutes, rinse with distilled water (do not pour off the stain first), then leave it to dry for microscopic examination.
The phagocytosis of sheep erythrocytes by macrophages and neutrophils in the mouse peritoneal cavity was observed, and the percentage of phagocytosis was calculated, i.e., the number of sheep erythrocytes phagocytosed per 100 phagocytes. The phagocytic index can also be expressed by dividing the total number of sheep erythrocytes phagocytosed in 100 phagocytes by 100, which is the phagocytic index. The phagocytic percentage and the phagocytic index are generally parallel.