Standard Operating Procedure for Testing Invasion and Endocytosis of Candida albicanson H4 Human Intestinal Epithelial Cells
Title or Type of Procedure: Invasion and Endocytosis of C. albicanson 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: Candida albicanshas been classified as a Biosafety Level 2 (BSL2) organism. Hazards for BSL2 organisms include 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.
1. Dilute an overnight C. albicansculture into fresh tissue culture medium to equal 2 x 105cells/ml.
2. Add 1 ml of the diluted C. albicansculture to each well containing epithelial cells.
3. Incubate at 37°C in a CO2incubator for 3 hrs.
4. Rinse cells with HBSS X 1.
5. Fix cells with 4% paraformaldehyde for 10 min.
6. Rinse cells with HBSS X 3 (2min washes).
7. Place coverslips atop piece of parafilm and pipet 80 microliters of primary antibody (1:100 dilution in 3% BSA) onto each coverslip. Cover with second piece of parafilm and make sure there are no bubbles/areas that are not in contact with antibody sln.
8. Incubate in humidified chamber at room temp for 1 hr.
9. Put coverslips back in appropriate well of tissue culture plate.
10. Wash cells for 2 min with HBSS X 3.
11. Place coverslips atop piece of parafilm and pipet 80 microliters of secondary antibody (1:2000 dilution in 3% BSA) onto each coverslip. Cover with second piece of parafilm and make sure there are no bubbles/areas that are not in contact with antibody sln.
12. Incubate in humidified chamber in the darkat room temp for 1 hr.
13. Put coverslips back in appropriate well of tissue culture plate.
14. Wash cells for 2 min with HBSS X 3.
15. Label slides and drop approx. 15-20 microliters (not too much!) of mounting medium onto slide (mounting medium is very thick – use a pipet tip with the end cut off). Avoid bubbles!
16. Dip coverslips in H2O and place onto slide, atop mounting medium (Fluoromount G, Southern Biotech).
17. Let dry overnight (in the dark) atop bench.
18. Seal with nail polish if needed.
Primary antibodies
(A) Rabbit anti-C. albicansIgG 1:100 dilution in 3% BSA (in ddH2O)
Biotin conjugated (BioDesign)
(B) Isotype control normal rabbit IgG1:100 dilution in 3% BSA
(Santa Cruz Biotechnology)
Secondary antibodies
(A) Streptavidin Alexa 568 1:2000 dilution in 3% BSA
(B) Goat anti-rabbit Alexa 568 1:2000 dilution in 3% BSA