Assessment of the Activation of Multiple Caspases in Macrophages

Abstract

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.

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....

Materials

List of materials used in this article

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