Standard Operating Procedure for Testing IL-8 Levels from H4 Cells
Title or Type of Procedure: Testing IL-8 Levels from H4 Cells
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: Potential for aerosolization during routine manipulation of Candidaalbicans and human H4 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. H4 cells are human intestinal epithelial cells, and work with human cells 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.
Paraformaldehyde is a toxic and hazardous chemical; extreme caution must be exercised when using paraformaldehyde to minimize risk of exposure. Personal Protective Equipment must be worn when handling paraformaldehyde.
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.
Day 1
1. Streak out C. albicansstrains to be used from -80C frozen stocks onto YPAD plates. Grow up overnight at 30°C
2. Seed each well of (2) 24-well sterile tissue culture plates with 1mL of H4 cells at a concentration of 1x10^5 cells/mL.
Day 2 or 3 (depending on when cells reach ~80% confluence)
3. Set up overnight C. albicanscultures in 2mL SDC at 30°C.
4. Feed H4 cells (aspirate and add 1mL fresh media per well)
Day 3 or 4 (cells confluent)
4. Dilute overnight C. albicanscultures into fresh tissue culture medium (Concentrations dependent on experiment) and seed each well with 100µl (media alone for control wells and media + Candida for experimental wells).
5. Incubate at 37°C with 5% CO2- 1 plate for 6hrs and the other for 12 hrs
6. Label 1.5 mL microfuge tubes with date and important corresponding well information (strain, yeast concentration, etc) for each of the 48 wells (2 – 24well plates)
6. At end of incubation time spin down each plate in centrifuge for 5 min at 1000rpm
7. Remove supernatant from each well and store in 1.5mL microfuge tubes @ -20°C