Standard Operating Procedure for Promega Cytotox 96ÒCell Damage Assay
Title or Type of Procedure: Promega Cytotox 96ÒCell Damage Assay
P. I. Cheryl Gale Lab Location: 13-125 PWB
Original Issue Date:01/31/2014
Revised Issue Date: 02/23/2017
Prepared By: Sara Gonia and Tim Heisel
Procedural Methods and Materials: See following sheets.
Hazard Identification and Risk of Exposure to the Hazards: Candida albicanshas been classified as a Biosafety Level 2 (BSL2) organism. Hazards for BSL2 organisms include potential for aerosolization during routine manipulation of C.albicans and human intestinal epithelial cells including transfer to test tubes and plates for growth, vortexing and/or centrifugation. C. albicansis not a respiratory pathogen and extremely large numbers of organisms are required to cause any infection so the risks to laboratory personnel are minimal. C. albicansare normal yeast organisms in most human GI tracts and cause disease only in immunocompromised individuals. Work with H4 cells, an established primary human intestinal epithelial cell line, and intestinal epithelial cells isolated from human fecal samples must be conducted in a certified biosafety cabinet. No food or drink is to be consumed in the laboratory. Quantities used are in the 0.1 - 2 µl range. Spill risk is negligible. Lab coats and gloves are worn at all times when handling yeast and yeast components (genomic DNA, protein). Any spills of yeast cultures or human cell cultures are to be decontaminated with at least 10% bleach by volume.
Exposure Controls Specific to Above Risk of Exposure: Use of protective wear including gloves and laboratory coats is required. Screw cap tubes and centrifuge lids are used to minimize aerosolization. No sharp objects will be used to transfer live cultures (no Pasteur pipets, no needles, etc). Follow lab disposal plan for all materials used in this protocol and lab decontamination plan for any spill. NOTE: Any immunocompromised individuals present in the lab must wear a custom-fitted N95 or equivalent respirator when working with C. albicansto avoid potential infections. All personnel requiring N95 respirators will be enrolled in the OHS respiratory protection program.
Waste Generated and Disposal Methods: Follow Gale lab SOP for disposal of waste.
Spill and Accident Response Procedures: Follow Gale lab SOP for any spills or accidents.
Notes: All laboratory personnel must take Bloodborne and Other Pathogens Training.
Procedure
1. Grow up epithelial cells in 100mL media in flat-bottomed 96-well plate. Be sure to include epithelial cells to enough wells so that everything can be done in triplicate (i.e. three wells for Target Maximum release, three wells for Target Spontaneous release and three wells for each strain used).
Day 1
2. From frozen stock, streak out C. albicansstrains onto YPD and incubate overnight at appropriate temp.
Day 2
3. Innoculate each C. albicansstrain in 1 mL fresh SDC media and incubate overnight with shaking at appropriate temp.
Day 3
4. Make 1:500 dilutions of each strain and determine cell concentration using hemocytometer. Equalize concentrations to 1.5 * 106.
5. Remove media from each well using aspirator.
6. Add 100mL fresh media or media containing candida to each well, as appropriate (Expirimental and Effector Cell Spontaneous Release wells will contain media+candida (1.5 *10^6 cell concentration): all other wells will contain media only).
7. Incubate for 7 hours 15 minutes at 37°C, 5% CO2.
8. Add 10mL lysis buffer to maximum release and volume correction wells.
9. Incubate for an additional 45 minutes. Check to make sure that cells in maximum release wells are lysed: if not, add another 5mL from each other well. Prepare assay solutions: Thaw assay buffer (at room temp or in 37C water bath), remove 12mL and store remaining buffer at -20C. Add the 12mL to bottle of substrate mix. Keep assay-substrate mixture in dark.
10. Centrifuge 250g for 4 minutes (about 1100-1200 rpm).
11. Transfer 50mL from each well (not containing target cells) into new plate. For other wells set up new cell dilutions: for Target Maximum Release wells 1:25 (2mL cells into 48mL media), for Target spontaneous release wells 1:5 (10mL cells into 40 mL media), and for experimental wells 1:10 (5mL cells into 45mL media) and add to new plate.
12. Add 50mL substrate mix to each well and incubate 30 min at room temperature in the dark.
13. Add 50mL stop solution to each well.
14. Read absorbance at 490 or 492nm.