Cell Phagocytosis Assay Protocol

• 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 are injected intraperitoneally with sheep erythrocyte suspension, and one hour later, the abdominal phagocytosis cells are dissected and collected, and the phagocytosis phenomenon on sheep erythrocytes can be observed by staining and microscopic examination. The phagocytosis function of the phagocytes could be determined by calculating the phagocytosis percentage or phagocytic index.

• 3 mL of 3% sodium thioglycolate Is aspirated with a sterile syringe and injected into the intraperitoneal cavity of the mice.
• Four days later, 1 ml of 1% sheep erythrocyte suspension Is injected into the intraperitoneal cavity of mice.
• One hour after the injection, the mice are killed by cervical dislocation, and the abdominal cavity is dissected and exposed. In the upper part of the abdominal cavity, the peritoneum is gently pinched with forceps, a small cut is made in the peritoneum, and 5 mL of pre-warmed PBS is injected into the peritoneal cavity with a pointed pipette, and the peritoneal cavity is rubbed repeatedly with the hand for about 1-2 minutes to flush out the phagocytes from the abdominal cavity of the mouse.
• Aspirate the peritoneal fluid with a sharp pipette and place it in a clean test tube.
• Blow the peritoneal fluid with a sharp pipette (try to avoid air bubbles), put a drop on a slide, and cover the slide for microscopic examination.
• To observe the results, the smear of peritoneal fluid can also be placed flat in a wet dish and incubated 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 drops 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.
• Observe the phagocytosis of sheep erythrocytes by macrophages and neutrophils in the mouse peritoneal cavity and calculate the phagocytosis percentage, 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.

Creative Bioarray Relevant Recommendations

• Based on superior expertise and extensive experiences, Creative Bioarray has developed a systematic approach to assess phagocytosis. These approaches are designed to help you analyze the function of specialized immune cells, evaluate the effects of antibodies and vaccines, as well as screen for TLR ligands, phagocytic activators, and inhibitors.

• Rub the peritoneal cavity sufficiently to wash down the phagocytes as much as possible.
• When aspirating the peritoneal fluid with a pointed pipette, avoid the abdominal organs as much as possible, to avoid damaging the blood vessels and causing bleeding, which will affect the experimental results.
• When staining with Rachel's stain, do not pour off the stain before rinsing, to avoid the fine particles in the stain adhering to the slide and affecting the clarity of the specimen.

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