Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from "Compendium Immunology Laboratory Guide".
Operation method
Selection and preparation of antigen-presenting cells Move Basic protocol for preparation of peritoneal macrophages by Streptococcus pyogenes stimulation Materials Listeria monocytogenes Sterile PBS H-2-compatible mice (see Table A.2A.1), including normal and Listeria monocytogenes-injected mice 3 weeks in advance D M E M medium in an ice bath. 3 % (V A O acetic acid 0.4% (m /V ) Taipan blue dye solution V D M E M-10 complete medium (room temperature and 37°C) 70 % (V7 V ) Ethanol IOml and 30m l syringes with 23 G needles Centrifuge Pipette microscopes 1 . Dilute Listeria monocytogenes with PBS in a 50 ml centrifuge tube to a suitable concentration, where 0.5 to 1.0 ml of the bacterial solution contains 5 to 10% of the LD50 (normally IO5 bacteria/ml for CBA/J or C57BL/6 mice and IO4 bacteria/ml for BALB/c mice). Cap the tube tightly and shake well. Draw up the diluted supernatant in an IOml syringe. 2 . Using a 23 G needle, inject 0.5 to 1.0 m l of bacterial suspension into the peritoneal cavity of H-2-compatible mice. Calculate the number of mice required for the preparation of sufficient cells according to step 6. Collect the abdominal cells after 3.7 to Ild (Unit 6.1). Collect the cells in an ice bath of DMEM to prevent macrophages from adhering to the wall of the tube. 4 . Place the collected cell suspension in a 50 ml centrifuge tube and centrifuge at 400~500 g for lOmin at 4°C. Collect the cells and resuspend them in 0.5 ml of pre-cooled DMEM. 5 . Aspirate a small amount of cell suspension and suspend 1:5 (WF) in 3% acetic acid solution to lyse the erythrocytes. Add an equal amount of Taipan blue stain and count the viable cells (see Appendix 3C, the cell count should be I X l O 7 to 2 X 107 cells). 6 . Add DMEM-10 complete medium to the centrifuge tube and adjust the cell concentration to 2X 106 cells/m l. If the serum concentration does not reach 8 % ~ 9 %, centrifuge the cells and discard the supernatant and dilute directly with DMEM-IO complete medium. If the serum concentration does not reach 8 % to 9 %, the cells can be centrifuged and the supernatant discarded and diluted directly with DMEM-IO complete medium. 7. Incubate at 37°C for 2 h or longer (but not overnight) to allow the macrophages to adhere to the wall. 8 . Blow the cells gently and repeatedly with a multichannel pipette, aspirate the suspended cells and add to preheated DMEM-IO Complete Medium at 37°C. Be careful to be gentle and not to touch the adherent cells. 9 . Examine the cells under a microscope. Isolated cells do not require further activation with IFN-7 if used within I d . This method allows for the preparation of a larger number of mouse peritoneal macrophages than the previous method, but requires an additional injection and a shorter period of time to collect the macrophages. The procedure is essentially the same as the basic protocol described above, except that 7-IOd after the initial bacterial injection, a single injection of I m l of autoclaved and sterilized 10 % (V/V) guinea pig peptone dissolved in water is administered directly into the peritoneal cavity of the mice. Cells were collected 3d later as described above (see Basic protocol, steps 3 to 9). 4 mice (e.g. C B A /J) Culture of Listeria monocytogenes (see module 6.6) Sterile PBS I O O m m Sterile Brain and Heart Agar Plate Aseptic brain and heart extract medium Bacterial cryopreservative: 20 % (V/V) glycerol dissolved in PBS, steam sterilized and stored at 4°C. 70um sterile nylon filter 50m l centrifuge tube Sterile bacterial applicator 37°C Incubator Aseptic Inoculation Ring 560n m spectrophotometer High-speed centrifuge and sterile capped tubes Sorvall R C-5 centrifuge with G S A rotor I m l freezing tubes 1 . Inject an appropriate amount of Listeria monocytogenes into the peritoneal cavity of 2 mice in order to obtain Listeria monocytogenes by the in vivo route and to ensure virulence. In the case of CBA/J mice, IO6 bacteria/mouse may be used. After 2 days, the mice are executed, the spleens are removed, and a suspension of splenocytes is prepared and passed through a 70 Mm sterile nylon filter and placed in a 50 ml centrifuge tube. Two additional mice are used as controls. Instead of in vivo passaging, Listeria monocytogenes clones can be picked directly from brain and heart agar plates and inoculated into brain and heart broth as in step 3. 2. Dilute the splenocyte suspension with PBS gradient (1 : 10, 1 : 100, 1 : 100) and place IOOm I of each group of diluted cell suspension on IOOm m m sterile Brain Heart Agar Plate, and spread the cells evenly with sterile bacterial spreader. The plates were incubated overnight at 37°C in an incubator. Count the number of clones per plate. Confirm that the coated control mouse splenocyte plates do not form clones and that the coated experimental mouse splenocyte plates form a large number of clones (10 to 100 clones per plate should be obtained for a 1:100 dilution of cells). 3 . Pick 4 typical clones from the plate with an inoculating loop and transfer each to 20 ml of sterile brain and heart medium (50 ml centrifuge tube). Note that the inoculating ring should be sterilized each time a clone is picked. Cover and mix. 4a. Preparation of same-day bacterial cultures: incubate tubes at 37°C with shaking every 30 m i n . After 3 h, a small amount of the culture is taken at regular intervals (e.g. every hour) and the OD values are measured spectrophotometrically. When all OD values reach 0.17, stop the incubation (about 4 h) and take a tube from it for re-amplification. 4b. Preparation of overnight bacterial cultures: The test tubes were incubated overnight at 37°C (no need to tighten the caps). On the second day, dilute the bacterial solution in the test tube with 1:1000 into sterile brain heart extract medium and incubate until the OD value reaches 0.5~1.0 (about 8 h). 5 . Store the culture solution at 4°C (0.5 ml each). If convenient, confirm the presence of Listeria monocytogenes in the culture the next day. In addition, Listeria monocytogenes can survive and amplify at 4°C. For overnight cultures, proceed to step 7. 6 . After screening, transfer 15 to 130 m l of brain heart extract medium containing bacteria to a sterile high-speed centrifuge tube, fix it in the sample rack of the shaker, incubate it at 37°C with slow shaking, check the OD 56c 5 value regularly, and stop incubation when the OD 56 value reaches 0.5 to L O (about 5 h). 7.700 g. Centrifuge the bacteria for 6 min. great care must be taken during this procedure: the culture contains a large number of bacteria. Suspend the bacteria in IOml of freezing solution. 8 . Detect the OD 56 value of the bacteria (can be detected by 1:20 dilution), and adjust the concentration of bacteria to I X l O7 ~ IO8 bacteria/ml (the amount of bacteria at OD 56 value of 1 is about 5 X 108 ). Bacterial fractions (0.1~Iml) can be stored at 80°C for more than 3 years. Use after each thawing, avoiding repeated freezing and thawing. No pathogenic bacteria are used in this protocol, but the rate of macrophage acquisition is low. Con A is dissolved in PBS (200ul/ml), filtered through a 0.22/xm filter, and stored at 1 20°C. The cells are then stored at 1 20°C in the laboratory. Each mouse is injected intraperitoneally with 500ul (IOOug) of Con A (Appendix 2E), and peritoneal macrophages are collected after 4d (see Basic Protocol, Steps 3 to 9). 10% (VAO L929 Cell Conditioned Medium (see Supplementary Scheme 2), dissolved in DMEM-10 Complete Medium 0.05% (V /V ) trypsin/0.53m m o l / L E D T A 100U/ml (lOng/ml) recombinant mouse IFN- 7 , solubilized in l ○ % L 929 conditioned medium 5m l syringe and 25 G needle I O O m m Sterile Petri dishes IOml sterile pipette 70p m sterile nylon filter Cell culture dishes and flasks (optional) 9 6-well flat-bottomed plates or 2 4-well plates or I O O m m cell culture dishes 1 . Mice are necropsied (Appendix 2G), the femur is removed and the muscle is debrided (Unit 6.1). 2. The femur is cut and the bone marrow is flushed out with 4 ml of 10 % L929 conditioned medium through a 5 ml syringe and a 25 G needle, and collected in sterile IOO m m cell culture dishes. 3 . Blow the cell mass repeatedly with a sterile IOml pipette and pass the cell suspension through a 70 Mm sterile nylon strainer. 4. Take a portion of the cell suspension and suspend it in 3 % acetic acid at 1 : 5 to lyse the erythrocytes. Add an equal amount of 0.4 % Taipan blue stain for counting. 4% Taipan blue staining solution was added to count the viable cells. Accordingly, the cell concentration was adjusted to IO6 cells/mL with 10 % L929 conditioned medium. 5a Expansion of cells: Cells were cultured in Petri dishes or flasks for 7d, digested with 0.05% (VAO) trypsin/0.53 mmol/L EDTA, collected and placed in 96-well culture plates at 5 X IO4~IO5 cells/well. 5b. Non-expansion culture: Place the cells in a culture plate: 9 6-well plates: 0.2 ml cell suspension/well 2 4-well plates: I m l cell suspension/well I O O m m Petri dishes: 12m l cell suspension/each 6. For expanded cells, continue to culture for 1~2d until the cells are confluent. For non-expanded cells, incubate for 5~7d with 10 % L 929 conditioned medium changed every 2 days. 7 . To enhance the efficiency of antigen presentation, the cells can be treated with recombinant mouse IFN-y (l0 ng/m l) dissolved in 10 % L929 conditioned medium for 48 h prior to the antigen presentation assay. Wall-cultured L 929 cells (A T C C cell line C C L L ) V Bacteria-free PBS, room temperature 0 . 0 5 % (W V ) trypsin/O.53 mmol/L EDTA V D M E M -IO complete medium 15m l centrifuge tube Sorvall RT- 6 0 0 0 Refrigerated tabletop centrifuge with HB-IOOO rotor culture flasks 0.45um filter membrane 1. Cultivate L929 cells in 25 cm2 flasks and aspirate the supernatant (10 ml) when the cells are in the logarithmic growth phase and growing at confluence. Add 3 ml of room temperature sterile PBS, shake the flask and let it stand for 1 min, shake gently once and then aspirate the PBS. 2. Add 3 ml of 0.5 % (V/V) PBS. Add 3m l of 0.5 % (V/V) trypsin/0.53 mmol/L EDTA and leave it for 5 min. Cover the bottle tightly and tap the bottle to dislodge the cells, or observe the cells under the microscope. 3 . Add IQml of DMEM-IO complete medium and transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 400~500 g for lOmin at 4°C. Aspirate and discard the supernatant, and then suspend the cells in DMEM-10 complete medium in the same amount as the original medium. 4 . Dilute the cell suspension 1:10 (WV) with DMEM-IO complete medium and transfer to appropriate sized culture flasks (0.4 ml cell suspension/cm2 flask). Continue incubation at 37°C until the cells are confluent, usually one week. 5 . Collect the cell supernatant, filter with 0.45p m filter membrane and dispense into 1~15m l portion each and freeze at 20°C. Spleen cell suspension in DME-IO complete medium (Unit 2.1) x/ D M E M -10 complete medium, 37°C LPS (e.g. Difco or Sigma) I O O m m Cell culture dishes 50 m l sterile centrifuge tube Sorvall R T - 6 0 0 0 refrigerated tabletop centrifuge with H B - I O O O rotor 75 cm2 cell culture flasks 1 . Remove the erythrocytes from the splenocyte suspension using ACK Erythrocyte Lysate (Unit 2.1). 1 ) to remove erythrocytes from the splenocyte suspension. 2 . Suspend the cells in D M E M -I O complete medium and count the viable cells (Appendix 3 0 , adjust the cell concentration to 5 XIO6 cells/ml. 3 . Transfer the cells to IOO m m cell culture dishes (IOml/dish) and incubate at 37°C for 2 h. After gently shaking the culture flask, transfer the suspended cells to a 50 m l centrifuge tube. 4 . At room temperature, centrifuge at 200-300 g for lOmin, aspirate the supernatant, resuspend the cells in pre-warmed DME-IO complete medium, count the cells (Appendix 30, adjust the cell concentration to 2 X 106 cells/m l.). 5 . Add LPS to a final concentration of 10 Mg/m l . Transfer the cell suspension to 75 cm2 cell culture flasks or IOO m m cell culture dishes and incubate at 37°C for 48 hours. 6 . Shake the flask gently to suspend the non-adherent cells, transfer the cell suspension to a 50 ml centrifuge tube, centrifuge at 200-300 g for lOmin, discard the supernatant, and resuspend the cells in DMEM-10 complete medium. 7. Centrifuge at 200-300 g for lOmin, aspirate the supernatant, resuspend the cells in pre-warmed DMEM-10 complete medium, count the cells, and adjust the cell concentration to 2 X 106 cells/m l. These cells can then be used as non-adherent cells. These cells can then be used as non-adherent antigen-presenting cells (cell 7.2). 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9 6-well culture plates
cells/ml).
Transfer the peritoneal cells to 96-well plates, IOUl/well (2 X 105 cells/well).
The cells were collected and placed in 9 6-well culture plates at 5 X IO4~IO5 cells/well.