Assessment of the Activation of Multiple Caspases in Macrophages

Overview

This video demonstrates the assessment of multiple caspase activations in bone marrow-derived macrophages through stimulation with the influenza virus. Following macrophage lysis, heat-inactivation of lysate, and centrifugation, intracellular caspases are collected for subsequent immunological analysis.

Protocol

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.

Category	Reagents	Preparation	Remarks	Safety Precautions
Media	L929 culture media	1) Heat inactivate FBS (see remarks). 2) Combine 43.5 mL of Iscove's Modified Dulbecco's Medium (IMDM), 5 mL of heat-inactivated fetal bovine serum (HI-FBS), 0.5 mL of sodium pyruvate, 0.5 mL of non-essential amino acids, and 0.5 mL of penicillin and streptomycin.	Heat-inactivate FBS by incubating at 56 °C for 30 min.
	Bone marrow-derived macrophage (BMDM) culture media	Combine 290 mL of IMDM, 150 mL of L929-conditioned media, and 5 mL of non-essential amino acids, 50 mL of HI-FBS, and 5 mL of penicillin and streptomycin.	Warm media to 37 °C in a water or bead bath before use. Media can be stored at 4 °C for up to 1 week.
	BMDM stimulation media with antibiotics	Combine 445 mL of Dulbecco's Modified Eagle Medium (DMEM), 50 mL of HI-FBS, and 5 mL of penicillin and streptomycin.	Warm media to 37 °C in a water or bead bath before use. Media can be stored at 4 °C for up to 1 week.
	BMDM stimulation media without antibiotics	Combine 450 mL of DMEM and 50 mL of HI-FBS.	Warm media to 37 °C in a water or bead bath before use. Media can be stored at 4 °C for up to 1 week.
	Media for infection (no HI-FBS)	Combine 495 mL of high-glucose DMEM with 5 mL of penicillin and streptomycin.	Warm media to 37 °C in a water or bead bath before use. Media can be stored at 4 °C for up to 1 week.
Sodium dodecyl sulfate (SDS) buffer	1 M Tris buffer (for 4x SDS buffer)	1) Dissolve 12.11 g of Tris in 80 mL of deionized water. 2) Adjust the pH to 6.8 by adding drops of HCl while stirring. 3) Add deionized water to adjust the final volume to 100 mL.	Buffer can be stored at 4 °C for up to 1 month.	HCl is caustic. Always wear appropriate personal protective equipment; this will include a lab coat and gloves. HCl should be handled in a fume hood.
	4x SDS buffer	1) Combine 60 mL of 1 M Tris (pH 6.8) solution, 24 mg of SDS, 100 mg of bromophenol blue, 120 mL of glycerol, 8.4 mL of β-mercaptoethanol (BME), and 90 mL of deionized water. 2) Stir on a warming stir plate to allow salts to go into the solution. 3) Add deionized water to adjust the final volume to 300 mL.	Buffer can be aliquoted and stored at −20 °C for up to 1 year.	SDS is caustic, and BME is carcinogenic. Always wear appropriate personal protective equipment; this will include a lab coat and gloves. SDS and BME should be handled in a fume hood.
Caspase lysis buffer	1 M 1,4-dithiothreitol (DTT)	Dissolve 0.154 g of DTT powder in 1 mL of phosphate-buffered saline (PBS). Allow DTT to go into the solution.	Buffer can be aliquoted into 200 μL aliquots and stored at −20 °C for up to 1 year.
	Caspase lysis buffer	Combine 35 mL of deionized water, 4 mL of NP-40, 750 μL of 1 M DTT, 1 protease inhibitor tablet, and 1 phosphatase inhibitor tablet.	Buffer can be stored at 4 °C for up to 1 month.
Radioimmunoprecipitation assay (RIPA) buffer	1.5 M Tris buffer (for RIPA buffer)	1) Dissolve 18.2 g of Tris in 80 mL of deionized water. 2) Adjust the pH to 8.8 by adding drops of NaOH while stirring. 3) Add deionized water to adjust the final volume to 100 mL.	Buffer can be stored at 4 °C for up to 1 month.	NaOH is corrosive and can cause burns. Always wear appropriate personal protective equipment; this will include a lab coat and gloves.
	10% (wt/vol) SDS solution (for RIPA buffer)	1) Add 10 g of SDS to 80 mL of deionized water. 2) Stir until dissolved. 3) Add deionized water to adjust the final volume to 100 mL.	The solution can be stored at room temperature for up to 6 months. If the solution precipitates, gently heat to re-dissolve.	SDS is caustic. Always wear appropriate personal protective equipment; this will include a lab coat and gloves. SDS should be handled in a fume hood.
	RIPA buffer (2x stock)	1) Combine 0.5 g of sodium deoxycholate, 1 mL of NP-40, 0.876 g of NaCl, 3.3 mL of 1.5 M Tris (pH 8.8), and 1 mL of 10% (wt/vol) SDS in 46 mL of deionized water. 2) Stir until dissolved. 3) Add deionized water to adjust the final volume to 50 mL.	2x RIPA stock can be stored at 4 °C for up to 1 month.	SDS is caustic. Always wear appropriate personal protective equipment; this will include a lab coat and gloves. SDS should be handled in a fume hood.
Sterile triggers	LPS	Dissolve lyophilized LPS from E. coli 0111:B4 in endotoxin-free water to a concentration of 5 mg/mL.	The solution can be stored at −20 °C for up to 2 years.
	0.5 M ATP	Dissolve 1 g of lyophilized ATP in 3.63 mL of endotoxin-free water.	The solution can be aliquoted and stored at −20 °C for up to 2 years.
Western blotting solutions	Running buffer (5x stock)	1) Combine 15 g of Tris base, 72 g of glycine, and 5 g of SDS in 900 mL of deionized water. 2) Stir until no particulates remain. 3) Add deionized water to adjust the final volume to 1 L.	Buffer can be stored at room temperature for up to 6 months.	SDS is caustic. Always wear appropriate personal protective equipment; this will include a lab coat and gloves. SDS should be handled in a fume hood.
	Transfer buffer  (10x stock)	1) Combine 30.3 g of Tris base and 72 g of glycine in 900 mL of deionized water. 2) Stir until no particulates remain. 3) Add deionized water to adjust the final volume to 1 L.	Buffer can be stored at room temperature for up to 3 months.
	Tris-buffered saline with Tween 20 (TBST)	1) Dissolve 2.42 g of Tris base and 8.76 g of NaCl in 850 mL of deionized water. 2) Adjust the pH to 7.4 by adding drops of HCl while stirring. 3) Add deionized water to adjust the final volume to 1 L. 4) Add 0.5 mL of Tween 20 (final concentration of 0.05% (vol/vol)).	Solution can be stored at room temperature for 1 month.	HCl is caustic. Always wear appropriate personal protective equipment; this will include a lab coat and gloves. HCl should be handled in a fume hood.
	5% (wt/vol) skim milk blocking solution	Dissolve 5 g of nonfat dried milk powder in 100 mL TBST.	The solution can be stored at 4 °C for up to 1 week.
Primary antibodies	Caspase-1 primary antibody	Dilute the anti-caspase-1 antibody 1:1,000 (vol/vol) by adding 10 μL of the antibody to 10 mL of 5% (wt/vol) skim milk blocking solution.	For optimal results, make fresh for each experiment. However, this solution can be used at least 3 times without loss of effectiveness. Store at −20 °C if reusing.
	Caspase-11 primary antibody	Dilute the anti-caspase-11 antibody 1:1,000 (vol/vol) by adding 10 μL of the antibody to 10 mL of 5% (wt/vol) skim milk-blocking solution.	For optimal results, make fresh for each experiment. However, this solution can be used at least 3 times without loss of effectiveness. Store at −20 °C if reusing.
	Caspase-3 primary antibody	Dilute the anti-caspase-3 antibody and the anti-cleaved caspase-3 antibody 1:1,000 (vol/vol) by adding 10 μL of each antibody to the same 10 mL of 5% (wt/vol) skim milk blocking solution.	For optimal results, make fresh for each experiment. However, this solution can be used at least 3 times without loss of effectiveness. Store at −20 °C if reusing.
	Caspase-7 primary antibody	Dilute the anti-caspase-7 antibody and the anti-cleaved caspase-7 antibody 1:1,000 (vol/vol) by adding 10 μL of each antibody to the same 10 mL of 5% (wt/vol) skim milk blocking solution.	For optimal results, make fresh for each experiment. However, this solution can be used at least 3 times without loss of effectiveness. Store at −20 °C if reusing.
	Caspase-8 primary antibody	Dilute the anti-caspase-8 antibody and the anti-cleaved caspase-8 antibody 1:1,000 (vol/vol) by adding 10 μL of each antibody to the same 10 mL of 5% (wt/vol) skim milk blocking solution.	For optimal results, make fresh for each experiment. However, this solution can be used at least 3 times without loss of effectiveness. Store at −20 °C if reusing.
	Caspase-9 primary antibody	Dilute the anti-caspase-9 antibody 1:1,000 (vol/vol) by adding 10 μL of the antibody to 10 mL of 5% (wt/vol) skim milk-blocking solution.	For optimal results, make fresh for each experiment. However, this solution can be used at least 3 times without loss of effectiveness. Store at −20 °C if reusing.
	HRP-conjugated β-actin primary antibody	Dilute the anti-β-actin antibody 1:5,000 (vol/vol) by adding 2 μL of the antibody to 10 mL of 5% (wt/vol) skim milk blocking solution.	This antibody should be prepared fresh for each experiment.
Secondary antibodies	Anti-rabbit antibody	Dilute the horseradish peroxidase (HRP)-conjugated secondary antibody 1:5,000 (vol:vol) by adding 2 μL of the antibody to 10 mL of 5% (wt/vol) skim milk blocking solution.	The antibody dilution should be prepared fresh each time.
	Anti-mouse antibody	Dilute the HRP-conjugated secondary antibody 1:5,000 (vol:vol) by adding 2 μL of the antibody to 10 mL of 5% (wt/vol) skim milk blocking solution.	The antibody dilution should be prepared fresh each time.
	Anti-rat antibody	Dilute the HRP-conjugated secondary antibody 1:5,000 (vol:vol) by adding 2 μL of the antibody to 10 mL of 5% (wt/vol) skim milk blocking solution.	The antibody dilution should be prepared fresh each time.

Materials

Name	Company	Catalog Number	Comments
0.45 μm filter	Millipore	SCHVU05RE
10 mL syringe	BD Biosciences	309604
12-well plate	Corning	07-200-82
18 G needle	BD Biosciences	305195
25 G needle	BD Biosciences	305122
50 mL tube	Fisher Scientific	50-809-218
70 μm cell strainer	Corning	431751
150 mm tissue culture dishes	Corning	430597
182-cm2 tissue culture flask	Genesee Scientific	25-211
ATP	InvivoGen	tlrl-atpl
BBL Trypticase Soy Broth	BD Biosciences	211768
Bead bath	Chemglass Life Sciences	CLS-4598-009
Biophotometer D30	Eppendorf	6133000010
Cell scrapers	CellTreat Scientific Products	229315
CO2 chamber	VetEquip	901703
Cuvettes	Fisher Scientific	14-955-129
Dissecting scissors	Thermo Fisher Scientific	221S
DMEM	Thermo Fisher Scientific	11995-073
Ethanol	Pharmco	111000200
Fetal bovine serum	Biowest	S1620
Filter paper	Bio-Rad	1703965
Forceps	Fisher Scientific	22-327379
Francisella novicida (U112 strain)	BEI Resources	NR-13
Gentamycin	Gibco	15750060
Heat block	Fisher Scientific	23-043-160
Herpes simplex virus 1 (HF strain)	ATCC	VR-260
High glucose DMEM	Sigma	D6171
Humidified incubator	Thermo Fisher Scientific	51026282
IMDM	Thermo Fisher Scientific	12440-053
Influenza A virus (A/Puerto Rico/8/34, H1N1 [PR8])	constructed per Hoffmann et al.
L929 cells	ATCC	CCL-1	Cell line for creating L929-conditioned media
L-cysteine	Thermo Fisher Scientific	BP376-100
MDCK cells	ATCC	CCL-34	Cell line for determining IAV viral titer
Microcentrifuge	Thermo Fisher Scientific	75002401
Non-essential amino acids	Gibco	11140050
NP-40 solution	Sigma	492016
PBS	Thermo Fisher Scientific	10010023
Penicillin and streptomycin	Sigma	P4333
Petri dish	Fisher Scientific	07-202-011
PhosSTOP	Roche	PHOSS-RO
Power source	Bio-Rad	164-5052
Protease inhibitor tablet	Sigma	S8820
Sodium hydroxide	Sigma	72068
Sodium pyruvate	Gibco	11360-070
Square Petri dish	Fisher Scientific	FB0875711A
Tabletop centrifuge	Thermo Fisher Scientific	75004524
Ultrapure lipopolysaccharide (LPS) from E. coli 0111:B4	InvivoGen	tlrl-3pelps
Vero cells	ATCC	CCL-81	Cell line for determining HSV1 viral titer