A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages
Published: October 31, 2023
Abstract
Source: Ball, B., et al. Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions. J. Vis. Exp. (2020)
In this video, we demonstrate a cytotoxicity assay to assess Cryptococcus neoformans, a pathogenic fungus, infection in macrophages. The cytotoxicity is determined by the release of cytoplasmic lactate dehydrogenase enzyme due to macrophage lysis caused by Cryptococcus neoformans infection.
Protocol
1. Verification of infection NOTE: Using a cytotoxicity assay, infection proficiency can be measured. The following protocol will highlight the application of a cytotoxicity product to measure LDH (lactate dehydrogenase) release. Other cytotoxicity products can also be used Preparation of C. neoformans infection of macrophage cells Preparation of fungal cells Using a glycerol stock, streak wildtype C. neoforma…
Declarações
The authors have nothing to disclose.
Materials
| 100 mM Tris-HCl, pH 8.5 | Fisher Scientific | BP152-1 | Maintain at 4°C |
| 60 x 15 mm Dish, Nunclon Delta | ThermoFisher Scientific | 174888 | |
| 6-well cell culture plate | ThermoFisher Scientific | 140675 | |
| Conical falcon tubes (15 mL) | Fisher Scientific | 05-539-12 | |
| Countess II Automated Cell Counter | ThermoFisher Scientific | AMQAX1000 | Not essential, haemocytometer can be used as an alternative. |
| CytoTox 96 Non-Radioactive Cytotoxicity Assay | Promega | G1780 | |
| DMEM, high glucose, GlutaMAX Supplement | ThermoFisher Scientific | 10566016 | |
| Fetal Bovine Serum (FBS) | ThermoFisher Scientific | 12483020 | Heat inactivate by incubating at 60°C for 30 minutes. Prepare 50 ml aliquots and flash freeze. Thaw prior to media preparation |
| Hemocytometer | VWR | 15170-208 | |
| HEPES | Sigma Aldrich | H3375 | Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly). |
| L-glutamine | ThermoFisher Scientific | 25030081 | Can be aliquot and frozen for storage. Thaw prior to media preparation. |
| Microcentrifuge | Eppendorf | 13864457 | |
| Penicillin : Streptomycin 10k/10k | VWR | CA12001-692 | Can be aliquot and frozen for storage. Thaw prior to media preparation. |
| Phosphate-Buffered Saline | VWR | CA12001-676 | Purchase not required. PBS can also be prepared but sterile filteration must be performed before use. |
| Pipette, Disposable Serological (10 mL) | Fisher Scientific | 13-678-11E | |
| Pipette, Disposable Serological (25 mL) Basix | Fisher Scientific | 14955235 | |
| Trypsin/Lys-C protease mix, MS grade | Pierce | A40007 | Maintain at -20 °C. |
| Urea | Sigma Aldrich | U1250-1KG | Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly). |
| Yeast-extract peptone dextrose broth | BD Difco | BM20 |
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Citar este artigo
A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages. J. Vis. Exp. (Pending Publication), e21660, doi: (2023).