A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages

Overview

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

1. Preparation of C. neoformans infection of macrophage cells
   1. Preparation of fungal cells
      1. Using a glycerol stock, streak wildtype C. neoformans strain (H99) onto Yeast-extract peptone dextrose (YPD) agar plate to isolate single colonies.
      2. Incubate overnight for 16 h at 37 °C in a static incubator.
      3. Select a single colony of wildtype C. neoformans strain and culture in 5 mL of YPD broth in a loosely capped 10 mL test tube. Perform in quadruplicate.
      4. Incubate overnight for 16 h at 37 °C in a shaking incubator at 200 rpm.
      5. The following day subcultures each overnight culture in a 1:100 dilution into 3 mL YPD broth.
      6. Measure Optical density (OD_600nm) values of fungal culture to determine the mid-log phase. Depending on the spectrophotometer, C. neoformans wild-type strain reaches the mid-log phase commonly following 6 to 8 h incubation in YPD with general OD_600nm values ranging from 1.0 to 1.5.
      7. Once cells reach the mid-log phase, take a 10 µL aliquot of fungal cell suspension into a clean 1.5 mL microcentrifuge tube and dilute 1:100 in sterile 1x phosphate-buffered saline (PBS). Count the number of cells using a hemocytometer.
      8. Collect and centrifuge cells at 1,500 x g for 10 min, gently wash the pellet with sterile room-temperature PBS, and repeat for a total of three washes.
      9. Resuspend cells in an antibiotic-free cell culture medium to achieve a concentration of 1.2 x 10⁸ cells/mL.
   2. Preparation of macrophage cells
      1. Visualize each well in the 6-well plate to ensure that cells have reached 70-80% confluence. Alternatively, cells can be measured to achieve approx. 1.2 x 10⁶ macrophage cells per well.
      2. Warm antibiotic-supplemented medium to 37 °C prior to cell culture work. Ensure the work environment is sterilized prior to cell culture work. Visualize cells using a light microscope to ensure cells are adherent and healthy.
      3. Remove the cell culture medium from the dish either with a serological pipette or a vacuum aspirator.
      4. Gently add 5-7 mL of sterile room-temperature PBS (phosphate-buffered saline) to the 60 x 15 mm dish.
      5. Gently tilt the dish to wash adhered cells.
   3. Co-culture of C. neoformans and macrophage cells
      1. Add 1 mL of the resuspended C. neoformans cells to 4 wells containing macrophage cells.
         NOTE: The number of plates required will need to be calculated prior to beginning the experiment. Add 1 mL of antibiotic-free medium to empty wells.
      2. Allow cells to incubate at 37 °C with 5% CO₂ for 3 h.
      3. Remove the cell culture medium from the plate either with a serological pipette or a vacuum aspirator.
      4. Gently add 1 mL of sterile room-temperature PBS.
      5. Gently tilt the plate to wash non-attached or non-phagocytosed extracellular C. neoformans cells.
      6. Add 1 mL of antibiotic-free medium to each well in the 6-well plate.
      7. Incubate cells at 37 °C with 5% CO₂.
   4. At selected time points (e.g., 1, 3, 6, 12, and 24 hours) collect supernatant for measurement of LDH release according to the manufacturer's instructions.
   5. At the same time points, uninfected macrophage cells will be lysed to a determined value for maximum cytotoxicity.
   6. Calculate cytotoxicity as follows:
      

Materials

Name	Company	Catalog Number	Comments
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