TITLE: Generating supplement-free conditioned media for proteomic analysis following human ovarian tissue culture.
AUTHORS: hannah.anvari, francesca.e.duncan duncan

[DESCRIPTION]
Purpose: This protocol is intended for use in generating supplement-free conditioned media for proteomic analysis. This protocol supplements the steps detailed in the processing and culture of human ovarian tissue for induced senescence as detailed in protocol dx.doi.org/10.17504/protocols.io.3byl4qbdjvo5/v2 

[GUIDELINES]
Guidelines for researchers working with human specimens
Researchers will adhere to all safety and training protocols required by the Northwestern Medicine/Northwestern University including but not limited to:

1.          Biosafety Certification
2.          Bloodborne Pathogens Certification
3.          Working with Formaldehyde Certification
4.          Collaborative Institutional Training Initiative (CITI program) certification


[STEPS]
SECTION: Wash steps to generate supplement-free conditioned media
1. Human ovarian tissue is processed and cultured according to an established protocol (dx.doi.org/10.17504/protocols.io.3byl4qbdjvo5/v2) upto Day 10. 
SECTION: Wash steps to generate supplement-free conditioned media
2. On day 10 of culture, two complete media changes are performed for each well as follows (Fig. 1): 
a.    In the original plate, each well is washed twice with pre-equilibrated basal media (MEM Alpha (1X) + GlutaMAX (Thermo, 32561037) without additional supplements).
b.    From the original plate, one transwell is removed at a time and released back into a wash dish containing pre-equilibrated basal media.
c.    The tissues are reloaded onto new transwells and moved into a new plate containing pre-equilibrated basal media (The plate map matches the original plate). One well is reloaded at a time.
d.    The new plate is reloaded into the incubator for an additional 24 hours.

 

SECTION: Wash steps to generate supplement-free conditioned media
3. When the culture period ends, all the conditioned media is collected into microcentrifuge tubes and snap-frozen over dry ice. The tubes are stored at -80°C until further use. 
SECTION: Wash steps to generate supplement-free conditioned media
4. The tissues from the inserts are collected by carefully peeling away the mesh bottom with sterile forceps. The edge of the transwell is held with curved forceps and the edge of the membrane is pierced with straight forceps such that it breaks away from the plastic edge. The remaining membrane is then peeled away from the edge.
SECTION: Wash steps to generate supplement-free conditioned media
5. The explants are then fixed according to an established protocol (dx.doi.org/10.17504/protocols.io.x54v9pyz1g3e/v1).
SECTION: Wash steps to generate supplement-free conditioned media
6. If freezing the tissues, the mesh bottom is placed into a dish of PBS and the tissues are carefully moved into a dry 1.5 mL microcentrifuge tube using a similar technique used to load the tissues onto the wells: take up some PBS and tissue (100-200 µL) into the cut tip of a 1000 µL pipette tip. Expel the contents into a dry 1.5 mL microcentrifuge tube and then remove excess PBS with a 200 µL pipette tip before placing them on dry ice. Store at -80°C.