STC-1 Passaging and PD Associated Microbe Stimulation Protocol

Cells are passaged when they reach approximately 70% confluence

Warm trypsin, 1XPBS, and complete medium in water bath for 15 min.

Aspirate off culture medium

Rinse cells with 10mL D-PBS

Add 3mL Trypsin to flask. Place flask back into 37°C/5% CO2 incubator for 7 minutes.

Observe cells under microscope to ensure they have detached from the bottom of the flask.

a. These cells are particularly sticky, so giving them a nice tap may be necessary to detach them.

Add 9mL of complete medium to flask and transfer cell suspension to a 15mL tube.

Spin down at 130g for 5 min

Aspirate off supernatant and resuspend cell pellet in 1mL culture medium.

Count cells

Prepare cell suspension at a concentration of 3x105 cells/mL

Add 1mL cell suspension per well in 12 well plate(s)

For PD associated microbe stimulation, allow cells to rest for approximately 24 hours,

Treat STC-1s with 10 colony forming units (CFU) of PD associated microbe per STC-1 cell

Incubate for desired time and then collect conditioned media and/or cells for downstream analyses

Following plating, allow cells to rest for 24 hours

Treat cells with 10ng/mL LPS for 24 hours

24 hr after LPS treatment, stimulate cells with 10 CFU of PD associated microbes per STC-1 cell

Incubate for desired time and then collect conditioned media and/or cells for downstream analyses