Feb 01, 2024
STC-1 Passaging and PD Associated Microbe Stimulation Protocol
- 1College of Medicine |University of Florida
Protocol Citation: Malu G Tansey 2024. STC-1 Passaging and PD Associated Microbe Stimulation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedmn7v47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 01, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94578
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Abstract
STC-1 Passaging and PD Associated Microbe Stimulation Protocol
Materials
Complete Medium
DMEM, high glucose, no glutamine
4mM L-glutamine
1mM sodium pyruvate
5% FBS
Other Reagents
0.05% Trypsin-EDTA
1X D-PBS without calcium and magnesium
75cm2 Flask
12 well plates
Passaging & Plating Protocol
Passaging & Plating 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
LPS Pre-Treatment
LPS Pre-Treatment
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