[PubMed] [Google Scholar] 26. quiescent cells did not increase, and in some cases a slight decrease was seen (as also noted by others). (B) The quality of the cell fractionation was assessed by immunoblotting for representative cytoplasmic (GAPDH) and nuclear (histone H2B) proteins. All lanes were leaded for comparative cell number. WC, whole cell extract; N, nuclear extract; C, cytoplasmic extract. Note the absence, in all cases, of GAPDH transmission in nuclear extracts, and a corresponding absence of H2B in cytoplasmic extracts. GAPDH transmission was increased in senescent cells, whereas H2B was not. No changes were seen between early passage growing and quiescent cells. mH2A, which we have previously shown to be increased in senescent cells  was also included for comparison. In the experiments shown in this physique, cultures designated as Early Passage were at passage 20, and those designated as Senescent were 1-2 passages prior to total cessation of proliferation. Quiescent cultures were prepared by serum deprivation (incubation of passage 20 cells in medium made up of 0.25% serum for 48 hr prior to harvest). Quiescence (>90% 2N DNA content) was confirmed by circulation cytometry. aging-03-955-s001.tif (811K) GUID:?9DA6C5AA-D4A4-4092-8A9D-A9BB06E5FC1E Supplemental Figure 2: Investigation of the parameters affecting the NanoOrange staining of whole cells During the development of the optimized protocol described in Methods, we diverse the relevant parameters to investigate their effect on the extent of staining and the robustness of the assay. (A) Effect of heat. The manufacturer’s Cot inhibitor-1 protocol recommends incubation at 95C for the optimal reaction of the dye with the protein. Incubation at 20C did not eliminate staining but decreased it significantly (* p < Cot inhibitor-1 0.01). The extent of staining was normalized to the 95C reaction. (B) Effects of chaotropic brokers. We investigated whether treatment prior to staining with a chaotropic agent, such with guanidium hydrochloride (Gu-HCl, 6M, 20C, 20 min) may increase the exposure of the proteins to the dye. Under our assay conditions this did not result in a measurable difference. The extent of staining was normalized to an comparative incubation made up of PBS only (without Gu-HCl). (C) Effect of time of incubation at 95C. The manufacturer's protocol suggest 20 min. In our assay varying the time between 15 and 25 min did not result in a measurable difference. The extent of staining was normalized to the reaction incubated for 20 min. (D) Effect of dye concentration. The manufacturer suggests diluting the dye answer provided in the kit to 2.5 l/ml, which they designate as the 1 x working concentration. We varied the concentration from 0.625 l/ml to 25 l/ml (40-fold range) and noted that this extent of staining was remarkably stable. There was a slight increase in staining between 0.625 l/ml and 6.25 l/ml, the values being statistically significant between 0.625 l/ml and 6.25 l/ml (p = 0.03) and 1.25 l/ml and 6.25 l/ml (p = 0.05) but not between 2.5 l/ml and 6.25 l/ml (p = 0.13). Staining reached an apparent plateau at 6.25 l/ml, which we chose as our standard protocol (this is 2.5 x the concentration suggested by the manufacturer for the staining of proteins in solution). The data shown in this panel were normalized to the reaction made up of 6.25 l/ml dye. In all panels the error bars designate the standard deviations. aging-03-955-s002.tif (1.6M) GUID:?5C629C6F-0799-43BB-99A2-CE58BE6BB75B Abstract Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to impact essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic Cot inhibitor-1 of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution Rabbit Polyclonal to KAL1 fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence,.
[PubMed] [Google Scholar] 26