Assessment of the Activation of Multiple Caspases in Macrophages

Published: January 31, 2024

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 Prepare L929-conditioned media. 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). Grow the cells in a humidified incubator at 37 °C with 5% carbon dioxide, CO2. After 7 days, collect the supernatant, and filter u…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
Assessment of the Activation of Multiple Caspases in Macrophages. J. Vis. Exp. (Pending Publication), e21905, doi: (2024).

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