Source: Han, J. et al., Evaluation of Caspase Activation to Assess Innate Immune Cell Death. J. Vis. Exp. (2023)
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.
1. Preparing the solutions
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.
3. Differentiating the BMDMs and plating for the experiments
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.
5. Collecting the combined supernatant and protein lysate to be used for caspase western blots
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. |
The authors have nothing to disclose.
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 |