Detection of phagocytic function of macrophages

Detection of phagocytic function of macrophages

Macrophages are responsible for phagocytosis, elimination of intracellular parasites, fungi, and the elimination of aging self-cells. It has an important role in specific humoral immunity or cellular immune response. Therefore, the phagocytosis and digestion function of macrophages is To a certain extent, it can reflect the body's immune status. There are two in vivo and in vitro methods for measuring the phagocytic function of macrophages, which are now introduced as follows:

1. In Vivo

(1) Materials

1. Mice are healthy Kunming mice, weighing 20 ~ 25g.

2. Growth liquid 05% hydrolyzed lactalbumin solution (prepared with Hanks solution, dissolved in heated water bath), add 10% inactivated calf serum, and adjust pH to 76 with 7% NaHCO3.

3. Bacterial suspension 4 mg / ml BCG vaccine (BCG) is diluted 8 times (ie 1 part BCG + 7 parts saline), or a 36-hour Streptococcus liquid culture can also be used.

(2) Method

1. The specific method of inducing large monocytes is: â‘  Anesthetize the mouse with ether (do not lethal), fix the limbs, disinfect the abdomen with 75% alcohol, lift the abdominal skin with ophthalmic forceps, cut the skin along the midline of the abdomen, and do not hurt the peritoneum Then separate the skin to both sides and fix it; â‘¡Inject 30ml of growth solution into the abdominal cavity with a 6-gauge needle and gently press on the abdomen to free the large monocytes.

2. Inject BCG vaccine about 5 to 10 minutes after the injection of growth fluid, then inject 1 ml of diluted BCG vaccine or Streptococcus culture 1 ml into the abdominal cavity, and gently press the abdomen of the mouse continuously, about 5 minutes later, use a 9-gauge needle Abdominal fluid was aspirated and added dropwise on a sterilized glass slide (flame sterilized before use), and then placed in a wet box. After incubation at 37 ° C for 30 min, large monocytes adhered to the slide, using physiological saline Or Hank's liquid rinse 1 or 2 times.

3. Staining and microscopic examination are resistant to acid staining. If the injection is not BCG, ordinary staining method can be used. After staining, check with oil lens.

(3) Results When inspected under the oil microscope, it can be seen that there are many phagocytized tubercle bacillus (BCG) or streptococcus in the cytoplasm of large monocytes.

Second, in vitro method (skin bubble method)

(1) Materials and methods

1. material

(1) 10% ethanol extract of cantharidin: take 10g of cantharidin, add 100ml of ethanol, and use it after leaching for several days at room temperature.

(2) Chicken red blood cell suspension: blood is collected from the inferior wing vein of the chicken or the heart, and stored in Alcohol at a ratio of 1: 5, and placed in a refrigerator at 4 ° C for 2 weeks. Take the required amount immediately before use, add physiological saline, centrifuge at 1 500r / min for 5min, wash 3 times, centrifuge at 2 000r / min for 5min for the third time, discard the supernatant, take the packed red blood cells and prepare a 5% suspension with saline .

2. method

(1) Skin bubble formation: the specific method is: â‘  cut the hair on the skin-scarred parts of the animal, such as the inner side of the thigh, the abdomen, etc., and shave it with a razor; , Affixed to the shaved area, press a small glass slide on the filter paper, then apply clean gauze on it, and fix it with adhesive tape or bandage; â‘¢ After 4 ~ 5h, the skin layer on the site of the cantharidin begins to move. At this time, the gauze can be removed , Slides and filter paper are removed together, change to an arched plastic cover, and fix it with gauze or tape to prevent the blisters from breaking; â‘£ After 48h, a blister of the same size as the cover slip is formed on the skin at the place of the spotted candida. Bacterial syringe carefully aspirate all the vesicle fluid, partially covered with sterile gauze, and the gauze can be removed after 2h.

(2) Phagocytosis test: 1ml of vesicle fluid plus 004ml of 5% chicken erythrocyte suspension, put it in a small test tube, and then place two small coverslips (cut from ordinary coverslips) on the bottom of the test tube. 37 ° C water bath for 30min, shaking every 10min. After the incubation, remove the small coverslip from the test tube (gently shake several times in the test tube liquid to free the free chicken red blood cells and non-adherent phagocytic cells from the slide), place it on the slide, blow After drying, stain with Wright's Giemsa's solution.

You can also centrifuge 1ml 1500r / min for 5 minutes, remove the supernatant, leave about 005ml of liquid, and then add 004ml of 5% chicken red blood cell suspension. After mixing, put in a 37 ° C water bath for 30min, shaking once in the middle. After incubation, the film was pushed, dried, stained, and examined microscopically.

(B) Results Observed the macrophage phagocytosis of chicken erythrocytes under oil microscope. There are 200 macrophages. The following two indicators can be used to indicate the phagocytic activity:

1. The percentage of phagocytosis is the number of macrophages that engulf chicken red blood cells per 100 macrophages.

2. The phagocytic index divides the total number of chicken red blood cells engulfed by 100 macrophages by 100 to obtain the phagocytic index, which is the average number of chicken red blood cells engulfed by each macrophage.

In addition, during counting, the degree to which chicken red blood cells are digested can also be recorded for reference. The degree of digestion is generally divided into the following four levels:

Level â… : undigested, cytoplasm light red or light yellow with green, and the nucleus is light purple.

Grade â…¡: Mild digestion, cytoplasm light yellow-green, nuclear condensation, purple-blue.

Grade â…¢: Severe digestion, pale staining of cytoplasm, and pale grayish yellow nuclei.

Grade â…£: Complete digestion. I see vacuoles with a shape similar to the size of chicken red blood cells. The edges are neat and the nuclei are blurred.

(3) Precautions In the phagocytosis test, adding a small amount of heparin to the vesicle fluid can prevent the test failure caused by fibrin precipitation. The method is easy to operate, does not require complicated instruments and equipment, and is convenient for promotion.

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