Griffin:Conditioned Medium Preparation
Protein secretion is a fundamental element to autocrine/paracrine biological functions. Protein secretion is transient, and can go to sub-ng/ml concentrations into culture media. Culture media contains salts, making selective precipitation of proteins a prerequisite for proteomics analysis. Non-secreted proteins (lysed cells) in culture medium can contaminate the secreted proteins preparations.
- Culture cells in appropriate size flask (T75, T125, or larger) under normal culture media (DMEM & FCS + penn/strep) until optimal confluency (The confluency for cells at changing to serum free media varies with each cell line, where preserving the phenotype while minimal cytoxicity or cell death is relevant; ~80-100%).
- Aspirate off the normal growth medium (FCS/penn/strep), wash monolayer 3x sterile PBS, then 3x in serum-free DMEM, and replenish cells with serum-free (SF) media. Preferable is serum- and phenol red-free DMEM (30 ml / T175 cm2 flask).
- ~48hours (or other optimal time point) incubation in SF media, pipet or pour off the conditioned media (CM) from the flask directly into a bottle top 0.22 micron filter while under vacuum, filter into a sterile bottle. Dispense 30 ml aliquots into sterile 50 ml tubes. Dispose of flasks containing cells.
- Spin CM in a high speed centrifuge at 100,000 xg for 45 minutes (24,000 RPM with SW-28 rotor)
- Take supernatant and precipitate CM proteins using ammonium sulfate: 0.561 g of ammonium sulfate/mL of CM (80% cut). Measure the total volume of clarified CM then add it to a beaker along with the ammonium sulfate.
- Stir in 4C room until ammonium sulfate dissolves, remove the stir bar, transfer the beaker into a ice bucket 6-12 hrs.
- Centrifuge the CM for 30 min at 37,000 x g (16,500 RPM with JA-17 rotor) at 4ºC.
- For large volumes, decant the supernatant after spinning and add additional CM to the same tube and repeat.
- Discard the supernatant and quick spin to remove any residual supernatant from the pellet.
- Resuspend the pellets in ice-cold PBS to a final volume of 2.5mL (note: the pellet may be very small or invisible. Keep track of where the pellet should be by marking the tube).
- Equilibrate a PD-10 Desalting column (Cat#17-0851-01 PD-10 columns desalt & recover macromolecules) with 25mL of ice-cold PBS.
- Apply 2.5mL of CM to the column. Discard flow-through.
- Elute with 3.5mL of PBS. Collect the eluate in a spin concentrator (10,000 MW cutoff, Amicon Ultra) and spin at high speed (3,200 x g) for ~15 minutes.
Ultrafiltration of CM for mass spectrometry is reproducible and appropriate for quantitative comparisons.
- Collect cell supernate & centrifuge at 1000g for 5 minutes (4°C) to pellet detached cells and large debris.
- Collect cell supernate & centrifuge for 1 hour at 100,000g (4°C) to pellet smaller debris and vesicles. Store @−80°C.
- CM supernatant ultrafiltration through centrifugation on an appropriate MW cutoff (MWCO) filter at 4000xg for 2 hours. Amicon High recovery centrifugal filters for concentrating samples.
Literature & Notes
- Precipitation with organic solvents (ethanol/acetone) requires ~7x volume solvent:aqueous, and may be prohibitive for large volume conditioned medium requirements for ng/ml abundance proteins.