scientificprotocols authored about 3 years ago

Author: Daniel T. O'Connor
  1. Can use 6- or 12-well tissue culture plates, although 6-well plates are probably better because the wells do not have to be 100% confluent to have plenty of cells per well to get good secretion counts (12- or 24-well plates are good for secretion studies using primary cultures).
  2. Consider Poly-L-Lysine or Poly-D-Lysine(Sigma) coating plates/wells if using a cell line that does not tightly adhere to the bottom (e.g., PC12 cells) and therefore may detach during the secretion study.
  3. Passage/seed cells onto plates to be used for secretion study 2 days before the study date for best results (we usually seed 2 days before, confluency 70-80%).
  4. For start of study: After removing old cell media, label cells with [3H]-L-norepinephrine ([3H]-NE). To label we add the[3H]-NE to pre-warmed (37oC) cell media to a final specific activity of 0.5-0.7 µCi/1 ml of media; we add 1 ml of[3H]-NE containing cell media to each well when using 6-well plates. Let the cells load in this media for about 2 hours to get good equilibrium of intra- and extracellular norepi pools.
  5. After the loading/labeling period remove the media and then wash cells 2x with pre-warmed calcium-containing secretion buffer (CaSB: NaCl 150 mM, CaCl2 2 mM, KCl 5 mM, HEPES 10 mM pH 7.4) by adding 1 ml to each well and then quickly (2-3 min. after adding the CaSB) aspirating it off.
  6. Place the cells in 1 ml (for 6-well plates) cell media (we use serum free DMEM ) for 30-60 min incubation to get rid of excess norepi (not taken up and stored in storage vesicles of cells). (Note: During this 30-60 min incubation period we usually get all of our secretion tubes prepared, containing the secretion buffer ± agonists, ± antagonists/modulators).
  7. Remove the cell media and wash the cells 2x as described in step #5 (may want to lengthen the wash times to 5 min per wash).
  8. Now add your pre-incubation solutions to the cells -- if you planned a preincubation/pre-secretion phase to expose the cells to agents before stimulating the cells to secrete -- or start the secretion phase immediately by adding the secretagogue containing solutions. We usually terminate our secretion phase within 15 minutes of initiation since very few agonists ever stimulate much secretion past this time.
  9. Terminate secretion by rapidly collecting the secretion buffer from each well into separate counting tubes, add 5 ml scintillation liquid, count. Add 1 ml of cell lysis buffer (CaSB + Triton 0.1%) to each well.
  10. After letting the lysis buffer stay in the wells for 10 min (gentle rotation), collect cell lysates in individual tubes, add 5 ml scintillation liquid, count.
  11. Calculate percent norepi secretion for each group (n = 3 cases/wells per group) using statistics package.


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