Cell Sample Preparation

Click here to download the protocol for cell sample preparation.

Cell Sample Preparation for Mass Spectrometry Analysis

Cell harvesting

Adherent cells. This protocol is for a plate that was seeded with 1×106 cells in 100-mm Petri dish before treatment. Biological triplicates (as a minimum) are recommended. The protocol is followed for each plate from start to finish (i.e., plates are not processed in parallel). After the desired time for growth and/or drug treatment, remove and discard the culture medium. Briefly rinse the cell layer with PBS to remove traces of medium. Add 1 ml of trypsinEDTA solution to the dish and examine cells using an inverted microscope to verify that the cell layer has been dispersed (usually within 2 – 5 min). Note: To avoid clumping, do not agitate the cells by hitting or shaking the plate while waiting for the cells to detach (For some cells it may be necessary to keep 37 °C to enhance detachment). Add 6 – 8 ml of complete growth medium and aspirate cells by gently pipetting up and down. Transfer cells and media to a 15-ml Falcon tube and centrifuge at 1500 rpm for 2 min. Remove and discard the supernatant and add 1 ml PBS to the cell pellet. Disperse the cell pellet by gently pipetting up and down two to three times using a 1-ml pipette. Transfer the cells and PBS to a 1.5-ml polypropylene tube with a cap. Centrifuge the cells at 2000 rpm for 2 min. Remove and discard the PBS supernatant.

Cells grown in suspension. No trypsin treatment necessary. Centrifuge cells in 15-ml Falcon tube as described above, remove and discard the supernatant and add 1 ml PBS to the cell pellet. Disperse the cell pellet by gently pipetting up and down two to three times using a 1-ml pipette. Transfer the cells and PBS to a 1.5-ml polypropylene tube with a cap. Centrifuge the cells at 2000 rpm for 2 min. Remove and discard the PBS supernatant.

Freezing

Snap-freeze the tube with the cell pellet in liquid nitrogen and store at -80 °C until analysis.

Submission to MS core

Contact Sammy Pardo (pardo@uthscsa.edu) or Dana Molleur (molleur@uthscsa.edu), to make arrangements to bring the tubes with the frozen cell pellets on dry ice to a designated location.

Protocol from Suryavathi Viswanadhapalli, Ph.D. and Ratna Vadlamudi, Ph.D.