Organ Conditioned Medium: Generating Medium from Harvested Organ Cells for Metastatic Cancer Studies

研究方案

1. Generation of Lung- and Brain- Conditioned Media

1. In a sterile tissue culture hood, invert PBS tubes containing lung or brain three times to remove residual blood from organs and aspirate PBS containing blood. Repeat with fresh cold PBS until solution appears clear with no evidence of blood.
2. Place lungs and brains in separate 60 mm2 glass petri dishes. Using two sterile scalpel blades, mince lungs or brains by repeatedly slicing back and forth until tissue fragments are approximately ~ 1 mm3 in size.
3. Determine the amount of media needed to resuspend tissue fragments from the calculated weight difference.
   NOTE: Lung and brain tissue are weight normalized by resuspension in a 4:1 media:tissue ratio (vol/wt).
4. Resuspend tissue fragments in appropriate volume (determined previously in step 1.3) of Dulbecco's Modified Eagle's Medium (DMEM):F12 media supplemented with 1x concentrated mitogen supplement and penicillin (50 µg/ml)/streptomycin (50 µg/ml).
5. Add resuspended lung or brain tissue fragments to one well of a 6-well plate.
6. Incubate tissue fragments in media for 24 hr at 37 °C and 5% CO2. Following incubation, collect conditioned media for each tissue and further dilute by adding three equivalent volumes of fresh media in a 50 ml conical tube.
7. Centrifuge at 1,000 x g in diluted conditioned media for each organ at 4 °C for 15 min to remove large tissue debris. Collect media supernatant and filter through a 0.22 µm syringe filter.
8. Pool conditioned media from each organ (i.e., lung with lung and brain with brain) from multiple mice to account for mouse-to-mouse variability. Aliquot and store conditioned media at -80 °C until use.

材料

Name	Company	Catalog Number	Comments
1x Phosphate-buffered saline	ThermoFisher Scientific	10010-023	Keep sterile
60 mm2 glass petri dishes	Sigma-Aldrich	CLS7016560	Keep sterile
DMEM:F12	Life Technologies	21331-020	Warm in 37 °C water bath before use, keep sterile
Penicillin-Streptomycin (10,000 U/ ml)	Life Technologies	15140-122	Keep sterile
50 ml conical tubes	Thermo Scientific (Nunc)	339652	Keep sterile
0.22 μm syringe filters	Sigma-Aldrich	Z359904	Keep sterile
6-well tissue culture plates	Thermo Scientific (Nunc)	140675	Keep sterile
Scalpel blades	Fisher Scientific	S95937A	Keep sterile