Suspended 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/ml for suspension 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.
⦁ Gently mix the cell suspension.
⦁ Remove an aliquot of cell suspension from the flask and count the cells using an appropriate cell
counting method.
⦁ Transfer the volume containing the desired number of cells to a new labeled 2.0 mL tube containing
80 mg zirconium beads. PLEASE FOLLOW THE RULES FOR LABELLING.
⦁ Centrifuge the cells at low speed (5 min, 500-800 g, room temperature) to gently pelletize the cells.
⦁ Aspirate and discard the culture medium (keep an aliquot back for medium analysis if desired).
⦁ Immediately and carefully add 1.8 mL warm blood bank saline 0.9% (37°C). Note:
Do not use PBS. Unbuffered saline helps remove residual media, waste products, cell debris, and phosphate salts.
Do not use pure water because pure water can cause osmotic shock and rupture cells.
⦁ Shake a little bit. DO NOT VORTEX.
⦁ Remove and discard all the solution.
⦁ To quench the cells, add 1 mL of pre-chilled (−50°C) 60% aqueous methanol supplemented with 70 mM HEPES
and vortex to ensure that all cells are covered.
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.