{"id":511,"date":"2021-08-11T10:41:43","date_gmt":"2021-08-11T15:41:43","guid":{"rendered":"https:\/\/wp.uthscsa.edu\/mass-spec\/?page_id=511"},"modified":"2021-08-11T11:28:26","modified_gmt":"2021-08-11T16:28:26","slug":"cell-sample-preparation","status":"publish","type":"page","link":"https:\/\/wp.uthscsa.edu\/mass-spec\/protocols\/cell-sample-preparation\/","title":{"rendered":"Cell Sample Preparation"},"content":{"rendered":"

[vc_row][vc_column width=”2\/3″][vc_column_text]Click here<\/a> to download the protocol for cell sample preparation.<\/p>\n

Cell Sample Preparation for Mass Spectrometry Analysis<\/strong><\/h3>\n

Cell harvesting<\/strong><\/p>\n

Adherent cells.<\/em> 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 \u2013 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 \u00b0C to enhance detachment). Add 6 \u2013 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.<\/p>\n

Cells grown in suspension.<\/em> 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.<\/p>\n

Freezing<\/strong><\/p>\n

Snap-freeze the tube with the cell pellet in liquid nitrogen and store at -80 \u00b0C until analysis.<\/p>\n

Submission to MS core<\/strong><\/p>\n

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.<\/p>\n

Protocol from Suryavathi Viswanadhapalli, Ph.D. and Ratna Vadlamudi, Ph.D.<\/em>[\/vc_column_text][\/vc_column][vc_column width=”1\/3″][\/vc_column][\/vc_row]<\/p>\n<\/div>","protected":false},"excerpt":{"rendered":"

[vc_row][vc_column width=”2\/3″][vc_column_text]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 […]<\/p>\n","protected":false},"author":159,"featured_media":0,"parent":450,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-templates\/child-page.php","meta":{"footnotes":""},"class_list":["post-511","page","type-page","status-publish","hentry"],"yoast_head":"\nCell Sample Preparation - Mass Spectrometry<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/wp.uthscsa.edu\/mass-spec\/protocols\/cell-sample-preparation\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Cell Sample Preparation - Mass Spectrometry\" \/>\n<meta property=\"og:description\" content=\"[vc_row][vc_column width=”2\/3″][vc_column_text]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. 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