Detection of phagocytosis and killing function of macrophages
Detection of phagocytosis and killing function of macrophages
An assay for macrophage phagocytosis and killing function. Author: J.E. Colligan et al, Translator: Xuitao Cao et al. This experiment is from "Compendium Immunology Laboratory Guide".
Operation method
Detection of phagocytosis and cytocidal function of macrophages Move Basic Program 1 Macrophage Phagocytosis Detection Material Monocytes/macrophages: e.g. macrophage cell lines, mouse peritoneal macrophages [Unit 6.1, 10 % required]. balanced salt solution (BSS) Bacteria (e.g., Listeria monocytogenes Li say Brain E G D ), overnight culture, live Normal serum, freshly isolated or freshly thawed and set aside on ice PBS with 5 % (v/v) FCS, pre-cooled on ice PBS containing 30 % (m A O sucrose (filtered for sterilization, stored at 4°C for several months) Diff-Quik stain (Baxter Healthcare) 10 mmX75 mm polypropylene or polystyrene plastic tubes with caps Slides and coverslips Oscillators (Labindustries) Cytospin 2 Cell Shaker (Shandon/Lipshaw) 1 . Add 100 ml of PBS, centrifuge at 250g for 2 min at 4°C, wash monocytes/macrophages, repeat the wash once and discard the supernatant . Resuspend the cells with B S S and adjust the concentration to 2.5X 107 cells/m l. Add 0. I m l of cell suspension in 10m m X 75m m plastic test tubes with lids. 2 . The Listeria monocytogenes suspension was mixed on a vortex shaker (about 2 X IO9 bacteria/ml) and diluted 10 times with B S S . For the detection of phagocytosis by macrophages, the bacteria should be thoroughly aspirated from the remaining bacterial medium before dilution with BSS. 3 . Mix the bacteria with sufficient shaking (to make a single bacterial suspension) and add 0.1 m l of the bacterial suspension to the capped test tubes described above (2.5 X I O7 bacteria and 2.5 X I O 6 cells per tube). 4 . Add 50M1 of normal serum pre-cooled on ice, then add B S S to a final volume of lml and cap the tubes tightly. Place the tube on an oscillator and oscillate at 37°C, 8r/m i n reciprocating for 20-30 min (not more than 30 min). 5a. Basic method: Centrifuge at 250 g for 8 min at 4°C, discard supernatant. Add 2 times the volume of ice pre-cooled BSS and gently mix the cells with a pipette. Repeat the washing of the cells twice to thoroughly remove any extracellular bacteria not phagocytosed by macrophages. Resuspend the cells with 5 % FCS/PBS pre-cooled on ice and adjust to the appropriate concentration (usually 2 ml of FCS/PBS is added to a cell concentration of 0.5 mg/kg). 5b. More rigorous method: Wash the cells as in step 5a, but finally resuspend the cells with I ml of ice-precooled BSS. I ml of PBS with 30 % sucrose is then added to the bottom of the tube with an I ml tip and centrifuged at 250 g for 8 min at 4°C. After carefully aspirating the PBS and sucrose, the precipitate is resuspended in ice-cooled 5 % FCS/PBS and the concentration adjusted to the appropriate level (usually around IO6 cells/ml after 2 ml). 6. Add 0.1 ml of cell suspension (about IXlO5 cells) into the flaker, centrifuge at room temperature, 650r/min for 5 min, and fix the cells onto the slide. For larger macrophages, the number of cells can be reduced as appropriate. 7 . Perform Diff-Quik staining as directed. More than 200 macrophages were examined under an oil microscope (enlarged 1000X) and the number of bacteria phagocytosed by each macrophage was counted and the phagocytic index was calculated: Bacteria (e.g., Listeria monocytogenes, Escherichia coli, or Staphylococcus), logarithmic growth phase. Frozen bacteria were inoculated in liquid medium and incubated overnight Balanced Salt Solution (BSS) Monocytes/macrophages: e.g., giant contempt cell lines, mouse peritoneal macrophages [Module 6.1, change to 10 % guinea pig peptone or 4 % (m A O sulphoacetate media], or human P B M C (Module 8.1) Normal serum, freshly isolated or freshly thawed and kept on ice BSS with 5 % (m A O normal serum, pre-cooled on ice Sterile water 2 m l cone-bottomed polypropylene plastic test tube with screw cap (Sarstedt) or IOmmX 75 mm cone-bottomed polypropylene plastic test tube with cap Labindustries Bacterial plates: e.g. TSA (tryptic soy agar) plates (Remel; store at 4°C, preheat to 37°C before use) 13 mmX IOOmm boracic acid glass tubes with screw-on lids (Fisher or Coring), sterile 1 . Mix overnight cultures of Listeria monocytogenes by shaking and dilute 300 times with BSS. Add the following ingredients to a plastic test tube with a cap (IOMM X 75 mM) or a plastic test tube with a screw cap (2 mL): 0.3 m l diluted bacterial suspension (2.5 X 106 bacteria) 50 ml normal serum, pre-cooled on ice Replenish B S S to a final volume of I ml If it is suspected that macrophages (e.g., human alveolar macrophages) may have engulfed other bacteria, an additional control group without Listeria monocytogenes should be set up. 2 . Tighten the cap of the test tube. Place the tubes on a shaker at 37°C for 15-20 min at 8r/m i n to allow macrophages to phagocytose the bacteria. Remove unphagocytosed extracellular bacteria by thorough washing (see basic protocol 1, step 5a or 5b) and resuspend the cells in I ml of BSS containing 5% serum. 3 . Take 4 screw-top glass test tubes and add 0.9 ml of sterile water to each tube. Add 0.1 ml of cell suspension (water lyses macrophages only) to the first tube and perform a 10-fold serial dilution in the remaining three tubes. Change the tip of the gun when diluting between tubes and mix the cells well. 4 . Mix the liquid in each tube well and apply 0.1 ml of each liquid onto a TSA bacterial plate preheated to 37 °C. 5 . Cover the remaining undiluted cell suspension tightly. Incubate at 37°C for 90-120 min to allow macrophages to kill the bacteria. The tubes were then placed on ice to inhibit bacterial growth. Incubate the cell suspension with 10-fold serial dilution and paint the plate as above. 6 . After the coated liquid is absorbed by the agar, the plates are incubated at 37°C for 24-28 h. Count the number of bacterial clones and compare the changes in the number of bacterial clones before and after incubation (e.g., after incubation at 37°C). Count the number of bacterial clones and compare the changes in the number of bacterial clones before and after incubation of the samples (if the number of bacterial clones decreases after incubation at 37°C, it means that the bacteria are killed by macrophages). For bacterial culture, a standard 37°C incubator was used instead of a CO2-saturated humidity cell incubator, which is too humid for bacterial culture. Pathogenic Listeria monocytogenes (strain A T C C :15313), or other bacteria, such as those isolated and cultured from patients Tryptone Broth (Difco) 70°C water bath (recommended) 1 . Inoculate the appropriate amount of bacteria into tryptone broth (the amount of inoculum depends on how the bacteria are stored, e.g., frozen, stored on semi-solid slant medium, or in broth), and incubate to logarithmic growth period (4 to 6 h) with shaking in a 37°C water bath. 2 . Add 0.5 m l of bacterial solution per portion to IO m m X 75 m m capped polypropylene plastic test tubes and store at 80°C for storage (can be stored for > 1 year). 4 . Early logarithmic growth of bacteria: Take Im l of the bacterial solution and add it to fresh broth, continue to incubate for 4~6 h. 5 . Heat inactivation of bacteria: Inactivate the bacteria in logarithmic growth stage in water bath at 70°C for 60 min. Centrifuge at 900 g for 20 min at 4°C and discard the supernatant. Add IOml of PBS to wash the bacteria. Finally, resuspend the bacteria with PBS and adjust the concentration to 1010 bacteria/day. For more product details, please visit Aladdin Scientific website.
guinea pig peptone or 4 % (m/V) sulphoacetate broth], or human PBMC (Unit 8.1)
bacteria or heat inactivation (see auxiliary protocols)
The concentration of cells was adjusted to the appropriate concentration (typically around IO6 cells/m l after 2 m l).
Phagocytic index = Percentage of positive phagocytes (>1 bacteria) X Average number of bacteria in positive cells
Basic Program 2 Macrophage Killing Function Assay Material
0.1 ml macrophage suspension (2.5 X 106 cells)
10m x n X 75m m Polypropylene or polystyrene plastic tubes with caps
Store at 80°C (for >1 year).
3 . Before use, thaw the bacteria and add a drop (approx. 30/xl) to 5 ml of bacterial medium (e.g. tryptone broth) using a pipette. Incubate overnight until the bacteria reach late logarithmic growth or plateau stage (about 2X 109 viable bacteria/ml).
Adjust the concentration to about 1010 bacteria/ml (2 X 109 bacteria/ml for overnight cultures).