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Optimization of redox metabolite detection in mammalian cells (part II)

ABSTRACT: Conditions were tested to optimize number of cells and extraction buffer for the detection of redox reactive metabolites from mammalian cells. Four different extraction buffers were compared. Derivatization of glutathion was explored as a condition as well.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Cultured Cells

SUBMITTER: Boryana Petrova

PROVIDER: ST001772 | MetabolomicsWorkbench | Thu Apr 22 00:00:00 BST 2021

REPOSITORIES: MetabolomicsWorkbench

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Project description:Four protocols were adapted and compared to enrich extracellular matrix proteins from mouse skin: 1. compartmental enrichment; 2. chemical decellularization; 3. enzymatic decellularization using phospholipase A2; 4. quantitative detergent solubility profiling (QDSP). All four protocols were started the same day, each using 35 mg of frozen back skin taken from the same initial back skin piece. The experiment was repeated three times starting from three different mice. Compartmental enrichment: skin samples were processed using the CNMCS commercial kit. Briefly, the skin tissue was homogenized in buffer C and successively incubated under agitation in buffers W, N, M and CS supplemented with the protease inhibitors from the kit. Extraction buffer C and insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Chemical decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, SDS/sodium deoxycholate, Triton X-100 and GuHCl. Extractions buffers as well as the final insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Enzymatic decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, phosopholipase A2/sodium deoxycholate. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. QDSP: skin samples were homogenized in sterile PBS and successively incubated in 3 buffers containing increasing concentrations in detergents (including SDS, sodium deoxycholate, NP-40) as previously described. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. For comparison with the four protocols described above, total skin lysates were also prepared for two of the mice. In this case, 20 mg of mouse skin were homogenized and incubated in a lysis buffer containing SDS, β-mercaptoethanol. Samples were centrifuged and the supernatant was kept aside, flash-frozen in liquid nitrogen immediately.

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Optimization of redox metabolite detection in mammalian cells (part II)

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