Assessment of the Activation of Multiple Caspases in Macrophages

1. Preparing the solutions

1. Prepare L929-conditioned media.
   1. Plate 1 × 10⁶ L929 cells (see Table of Materials) in an 182 cm² tissue culture flask containing 50 mL of L929 culture media (see Table 1 for the preparation of the media).
   2. Grow the cells in a humidified incubator at 37 °C with 5% carbon dioxide, CO₂.
   3. After 7 days, collect the supernatant, and filter using a 0.45 µm filter. Prepare 50 mL aliquots (store frozen aliquots at −80 °C for up to 1 year).
2. Prepare 500 mL of bone marrow-derived macrophage (BMDM) culture media (Table 1).
3. Prepare 500 mL of BMDM stimulation media (Table 1).
4. Prepare 500 mL of media for infection (Table 1).
5. Prepare 100 mL of 1 M Tris buffer (Table 1).
6. Prepare 4x sodium dodecyl sulfate (SDS) buffer (Table 1).
7. Prepare 1 mL of 1 M 1,4-dithiothreitol (DTT, Table 1).
8. Prepare 40 mL of caspase lysis buffer (Table 1).
9. Prepare 100 mL of 1.5 M Tris buffer (Table 1).
10. Prepare 100 mL of a 10% (wt/vol) SDS solution (Table 1).
11. Prepare 50 mL of a 2x radioimmunoprecipitation assay (RIPA) buffer (Table 1).
12. Prepare a 5 mg/mL lipopolysaccharide, LPS solution (Table 1).
13. Prepare a 0.5 M adenosine triphosphate, ATP solution (Table 1).
14. Prepare the western blotting solutions.
    1. Prepare 1 L of 5x running buffer stock (Table 1).
    2. Prepare 1 L of 10x transfer buffer stock (Table 1).
    3. Prepare 1 L of Tris-buffered saline with Tween 20 (TBST, Table 1).
    4. Prepare 100 mL of a 5% (wt/vol) skim milk blocking solution (Table 1).
15. Prepare the primary antibody solutions.
    1. Prepare 10 mL of caspase-1 primary antibody (Table 1).
    2. Prepare 10 mL of caspase-11 primary antibody (Table 1).
    3. Prepare 10 mL of caspase-3 primary antibody (Table 1).
    4. Prepare 10 mL of caspase-7 primary antibody (Table 1).
    5. Prepare 10 mL of caspase-8 primary antibody (Table 1).
    6. Prepare 10 mL of caspase-9 primary antibody (Table 1).
    7. Prepare 10 mL of HRP-conjugated β-actin primary antibody (Table 1).
16. Prepare the secondary antibody solutions.
    1. Prepare 10 mL of anti-rabbit secondary antibody (Table 1).
    2. Prepare 10 mL of anti-mouse secondary antibody (Table 1).
    3. Prepare 10 mL of anti-rat secondary antibody (Table 1).

2. Isolating bone marrow-derived macrophages

NOTE: For this protocol, 6-10-week-old wild-type mice with intact programmed cell death (PCD) pathways or mutant mice with the PCD regulators, effectors, or molecules of interest deleted or altered can be used.

1. Euthanize a mouse in a CO₂ chamber with a flow rate that displaces 10%-30% of the cage volume per min for 2-3 min. Then, perform a secondary euthanasia method, such as cervical dislocation. Follow all additional facility-, institution-, and government-specific guidelines and regulations wherever applicable.
2. Dissect the mouse to retrieve the hind leg bones.
   1. Pin the mouse on its back so the abdomen is exposed. Spray with 70% (vol/vol) ethanol to sterilize the hind legs and abdomen.        
      CAUTION: Ethanol is flammable. Keep it away from open flames.
   2. Use scissors to make an incision at the midline of the abdomen; continue cutting toward the legs to make the femurs visible.
   3. Take the right rear leg and pull the skin away from the body toward the midline. Detach the leg from the body by severing the adductor muscles toward the midline; then, cut the leg between the hip joint and the spine. Next, cut the paw off distal to the ankle, and remove excess tissue from the bone by peeling off the skin and using slightly opened scissors to strip off the calf tissue.
   4. Place the leg on a 70% (vol/vol) ethanol-soaked towel and dissect the tibia and the femur.
      1. Clasp the tibia and femur each with a separate set of forceps. Gently press the tibia against the knee joint's natural direction; this will cause the tibia to break at the knee.
      2. Use the forceps and dissecting scissors if needed to remove any remaining hanging tissue. Save the tibia for later use by placing it on the ethanol-soaked towel.
      3. Collect the femur in the same way, by snapping off the knee.
   5. Repeat steps 3 and 4 above for the left leg to remove it from the body and dissect the tibia and femur.
   6. Spray the bones with 70% (vol/vol) ethanol.
   7. Clean off the bones by placing them on a clean 70% (vol/vol) ethanol-soaked towel, squeezing the fleshy part in the towel, and rubbing the towel against the bone to remove excess tissue.
3. Once both femurs and both tibias are clean, spray all four bones with 70% (vol/vol) ethanol. Collect the bones in a sterile Petri dish and rinse them with 10 mL of BMDM culture media by gently swishing the media in the dish.
4. Fill a 10 mL syringe with 10 mL of fresh BMDM culture media and attach a 25 G needle.
5. Pick up the tibia using forceps; then, cut the ankle joint at a ~45° angle.
6. Flush the bone marrow from the tibia.
   1. Hold the tibia over a 50 mL tube, with the narrow end of the tibia pointing down. Dispense the media over the tibia from the filled syringe.
   2. Insert the needle (gently at first) at the top end of the marrow and dispense the media.
   3. Remove the needle and then insert again. Use short, high-pressure pushes to dispense the media and move the needle into the marrow.
   4. Once the media begins to flow out from the bottom of the bone, use short, high-pressure pushes to continue to flush the cells out. Monitor the color of the bone during this process, and when the bone is white, discard it.
   5. Do this for both tibias.
7. Repeat steps 5 and 6 above for the femurs, cutting at the hip joint as the tibia was cut at the ankle joint. Use the same 50 mL tube to collect the BMDM culture media as the marrow is flushed.
8. Once all four bones have been flushed, aspirate the marrow and media from the 50 mL tube up and down 3x through an 18 G needle on a 10 mL syringe, rinsing the sides of the tube each time to disperse the marrow.
9. Adjust the final volume in the 50 mL tube to 30 mL with BMDM culture media, and ensure the cells are thoroughly suspended.
10. Use a 70 µm cell strainer to filter the BMDM culture media from the 50 mL tube.
11. Plate the resulting cell suspension, which contains the bone marrow progenitor cells, into three 150 mm tissue culture dishes by adding 10 mL (or ~20 × 10⁶ cells) to each. Then, add an additional 10 mL of BMDM culture media to each dish. Incubate in a humidified incubator at 37 °C.

3. Differentiating the BMDMs and plating for the experiments

1. Incubate the plated bone marrow progenitor cells at 37 °C for 3 days. Then, remove each dish, and add an additional 5-8 mL of BMDM culture media (Table 1). Return to the incubator at 37 °C.
2. On day 5 after the initial plating, remove each dish, and add an additional 5 mL of BMDM culture media. Return to the incubator at 37 °C.
3. On day 6, remove each dish, and discard the media. Then, add 10 mL of cold (stored at 4 °C) phosphate-buffered saline (PBS) to wash once. Discard the 10 mL of cold PBS wash. Then, add 10 mL of fresh, cold PBS to each dish, and incubate each dish on ice for 5 min.
4. Using a cell scraper, gently scrape the cells from all three dishes into one 50 mL tube. Gently spin down the cells at 270 × g at 4 °C for 5 min; then, discard the supernatant.
5. Add 20 mL of BMDM culture media, pipette up and down to resuspend the pellet, and count the cells.        
   NOTE: It is expected that each mouse will yield approximately 60 × 10⁶-100 × 10⁶ cells.
6. Plan out the 12-well plate layout for the desired in vitro cell death/inflammation stimulation assay. Plan to plate 1 × 10⁶ cells per well. For each planned stimulation, include at least three biological replicates, and plate one set of wells to be harvested in caspase lysis buffer and a second set of wells to be harvested in RIPA buffer.
7. Plate 1 × 10⁶ cells in 1 mL of BMDM culture media per well in 12-well plates. Culture overnight in a humidified incubator at 37 °C before proceeding to the cell death/inflammation evaluation. After overnight incubation, remove the media, and add 1 mL of warm (37 °C) PBS to each well to wash the cells.
8. Remove the PBS wash and add 500 µL of BMDM stimulation media with antibiotics (if performing non-bacterial stimulations) or BMDM stimulation media without antibiotics (if performing bacterial stimulations) (Table 1). Incubate for 2 h before moving to step 4 for in vitro stimulation/infection.

4. Stimulating or infecting the cells

CAUTION: The agents included in this protocol are potentially pathogenic and should be handled with the appropriate precautions in a biosafety level 2 (BSL2) facility with approval from the relevant institutional and governmental authorities.

1. Stimulate the BMDMs to activate cell death with the trigger of interest.
   NOTE: For the purposes of this protocol, influenza A virus (IAV), herpes simplex virus 1 (HSV1), Francisella novicida, and LPS + ATP are used for illustration, but other triggers can be used.
   1. Example stimulation 1: Infect with IAV (A/Puerto Rico/8/34, H1N1 [PR8]) (constructed as per Hoffmann et al.; the viral titer for the multiplicity of infection [MOI] determination is calculated by a plaque assay in MDCK cells):
      1. Calculate the volume of virus needed for infection at a multiplicity of infection (MOI) of 20 plaque-forming units (PFU) using equation (1) and equation (2):
         
         
         
   2. Remove the media from the BMDMs and wash the cells once with 500 µL of PBS. Add 450 µL of IAV (20 MOI) in high-glucose DMEM without heat-inactivated fetal bovine serum, (HI)-FBS to each well. Incubate the plates at 37 °C for 1 h in a humidified incubator to allow absorption.
   3. Remove the plates and add 50 µL of HI-FBS. Return the plates to the incubator at 37 °C. Incubate for a total of 12 h.

5. Collecting the combined supernatant and protein lysate to be used for caspase western blots

1. After 4 h, 12 h, or 16 h of incubation (the specific timing is dependent on the trigger used), remove the plate from the incubator.
2. Remove 150 µL of the supernatant; discard or save this for other supernatant analyses (e.g., enzyme-linked immunosorbent assay [ELISA]). Do not remove the remaining supernatant.
3. Create the protein collection solution by combining 50 µL of caspase lysis buffer + 100 µL of 4x SDS buffer per well (Table 1). Then, add 150 µL of the mix to each well.
4. For each well, pipette the mixture up and down to collect the lysed cells and supernatant. While pipetting, also scrape the bottom of the well with the pipette tip to disrupt the cells. After scraping and pipetting, collect the protein lysate into labeled 1.5 mL tubes.
5. Use a heat block to heat all the tubes to 100 °C for 12 min.
6. Pellet any insoluble components by centrifuging at 14,500 × g for 30 s at room temperature.
   ​NOTE: This is a pause point – the protein from the combined supernatant and protein lysates can either be used immediately or stored at −20 °C for up to 2 months or at −80 °C for up to 6 months until ready to use.