Griffin:Conditioned Medium Preparation

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(New page: ==Overview== ==Protocol== *Culture cells in appropriate size flask (T75, T125, or larger) under normal culture media (DMEM & FCS + penn/strep) until optimal confluency (The confluency f...)
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==Overview==
==Overview==
 +
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.
==Protocol==
==Protocol==
*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%).
*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.
-
*Aspirate off the normal growth medium (FCS/penn/strep), wash monolayer 3x sterile PBS, and replenish cells with serum-free (SF) media.
+
===Ammonium Sulfate===
-
Ideally cells will be 100% confluent with out too many dead or lysed cells at the time of
+
*Spin CM in a high speed centrifuge at 100,000 xg for 45 minutes (24,000 RPM with SW-28 rotor)
-
harvesting.
+
*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.
-
After ~48hours incubation in SF media, cells and the conditioned media (CM) are collected as
+
*Stir in 4C room until ammonium sulfate dissolves, remove the stir bar, transfer the beaker into a ice bucket 6-12 hrs.
-
follows.
+
*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.
-
Sterile-filter conditioned media by pouring it 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.
+
===Ultrafiltration===
-
Before collection of the conditioned media (CM),
+
*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===
 +
 
 +
http://www.ncbi.nlm.nih.gov/pubmed/17464941
 +
 
 +
 
 +
 
 +
Ultrafiltration of CM for mass spectrometry is reproducible and appropriate for quantitative comparisons.

Revision as of 16:07, 20 February 2013

Contents

Overview

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.

Protocol

  • 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.

Ammonium Sulfate

  • 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

  • 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

http://www.ncbi.nlm.nih.gov/pubmed/17464941


Ultrafiltration of CM for mass spectrometry is reproducible and appropriate for quantitative comparisons.

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