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…
Offenlegungen
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 |
Tags
Diesen Artikel zitieren
Assessment of the Activation of Multiple Caspases in Macrophages. J. Vis. Exp. (Pending Publication), e21905, doi: (2024).