Adherent Cell Procedure
Review Tissue handling precautions before proceeding.
This is a general procedure and the results can vary according to cell type. In general, a range such as 5×106
up to 1×107 cells (Note: cells/cm2 for adherent cells) will be suitable,
but this is solely dependent on the cell type in question. It is always imperative to run small trials to optimize for your case.
⦁ For control purposes, please make blank medium which must be incubated without cells.
⦁ Carefully remove all supernatant from the cells (keep an aliquot back for medium analysis if desired).
⦁ Wash cells quickly with blood bank saline 0.9% at 37°C (with enough volume to fully cover the cellular monolayer)
at room temperature. Do not use PBS. Unbuffered saline helps remove residual media, waste products,
cell debris, and phosphate salts. Do not use pure water because it can cause osmotic shock and rupture cells.
⦁ Remove all blood bank saline 0.9% by pipetting carefully until no solution remains.
Note: If your cells are weakly adherent, it is imperitative to
use extra care to not disturb the cells, as this will result in biomass loss.
⦁ To quench the cells, add 1 mL of pre-chilled (−50°C) 60% aqueous methanol supplemented with 70 mM HEPES
then agitate the flask to ensure that all cells are covered.
Note: the volume should have been previously optimized to be enough to fully cover the cellular monolayer.
⦁ Use a disposable cell scraper to remove all the cellular monolayer from the flask surface.
⦁ Harvest the quenching solution/cell mixture using a disposable pipette and transfer to a labelled Eppendorf
or Falcon tube (depending on volume) previously labeled. PLEASE FOLLOW THE RULES FOR LABELLING.
⦁ Proceed to the extraction step or store snap frozen in LN2 and store at −80° C until analyzed
and, if needed, all transportation should be carried out in dry ice.