Hoechst 33342 nuclear stain

For cells expressing GFP fusion protein, Hoechst 33342 or Draq5 nuclear stain is added to the fixative to ready the cells for image acquisition. For immunofluorescence staining, the cells are incubated with PBS-TB (PBS with 0.2% Triton X-100, 0.1% bovine serum albumin [BSA]) for 10 min to permeabilize the cell membranes. Primary antibody is then added at 0.5 to 5 pg/mL in PBS-TB and incubated at room temperature for 1 hr or at 4°C overnight. The wells are then washed two times with PBS-TB. Fluorescently labeled secondary antibody is added at 2 pg/mL together with 10 pg/mL Hoechst 33342 nuclear stain in PBS-TB and incubated at room temperature for 1 to 2 hr. The cells are then washed with PBS-TB and once with PBS before image acquisition. 

Fig. 12 The method of development of A647 MSN-RGD-PdTPP (reproduced with permission from Ref. 92) (a) and visualised in MCF-7 cells where blue represents Hoechst nuclear stain and red the nanoparticles (b). Image reproduced with permission from Ref. 92.

Figure 10 HCS-enabled screening of gap junctions - Rat glioma C6 cells stably expressing gap junction proteins are stained with Hoechst 33342 dye (red nuclear stain, Aex = 365 nm) and mixed with a small population of the same cells that had also been stained with a cytoplasmic dye, calcein AM (green, iex = 480 nm). After 1 h, the cells have settled and the two populations are generally easily distinguished (A). Only a few of the non-calcein dyed cells have picked up any calcein. After 2 hr of incubation (B), the calcein has spread extensively throughout the culture and this change in the distribution of dye is easily imaged on the ArrayScan II...

Nuclear stains (i.e., Hoechst, DAPI) Transverse or pulse field electrophoresis Assessment of Soluble DNA Flow cytometric analysis of DNA content... 

Fig. 2 Fluorescence microscopy of complex (1), denoted Ir(ppy)2(pybz) in CHO cells published by Williams et al The complex was co-locahsed with a nuclear stain (Hoechst). Images were acquired without a delay between pulse and acquisition (left) and with a delay (right). 

Cell number frequency distribution of nuclear DNA content of cell population, protein content, protein synthesis (e.g., 14C-labeled methionine incorporation), DNA synthesis (e.g., tritiated thymidine incorporation), DNA stains (e.g., Hoechst 33 342 4, 6-diamidino-2-phe-nylindole, DAPI picogreen) mass tracker dyes (e.g., LysoTracker green for lysosomes, MitoTracker deep red for mitochondria, ER-Tracker blue-white DPX for endoplasmic reticulum)... 

Hoechst 33342 (trihydrochloride, trihydrate 10 mg/ml) from Molecular Probes, Eugene, OR, to stain nuclei (cell-permeant nuclear counterstain). 

Fig. 4. Combined use of flow cytometry/cell sorting and confocal laser scanning microscopy. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine and then analyzed for mitochondrial membrane potential and NAD(P)H fluorescence (A). Cells in different regions of the cytogram were then sorted, and subsequently stained with the DNA fluorochrome Hoechst 33342. These cells were then examined by CLSM. (B), (C), and (D) show three-dimensional reconstructions of nuclei from cells sorted from healthy, early apoptotic, and late apoptotic populations. Whereas healthy cells show normal round nuclei, early and late apoptotic cells show progressive chromatin condensation/margination and nuclear fragmentation.

Fig. 8.4 Schematic diagram illustrating methods for quantifying the subcellular distribution of plasmid DNA (pDNA). After the transfection of rhodamine-labeled pDNA, the endosome/lysosome fraction and nuclear fraction was stained with LysoSenser DND-189 and Hoechst 33342, respectively to discriminate the subcellular localization of pDNA. For the data analysis, the pixel areas of each cluster on plasma membrane, S (mem), endosomes/lysosomes, Sj(end/lys), cytosol s, (cyt) and nucleus S (nuc) were separately summed in each XY-plane, and are denoted as S2 j(mem), S2 j(end/lys), S2 j(cyt) and S2 j(nuc), respectively. The values of S2 j(mem), S2 j(end/lys), and S j(nuc) in each X-Y plane were further summed and are denoted as...

Nuclear-associated vesicles (either chromatin-bound or fused into an envelope) can be labeled. Alternatively, vesicles can be prelabeled prior to nuclear assembly reactions. To label chromatin-associated vesicles, a 10-/il extract containing nuclei is mixed with 10 ju.1 of DiOC6 or DilCis, each at 20 /xg/ml in egg lysis buffer (stock B), and incubated at room temperature for 20 min. Samples are visualized under the fluorescence microscope using the appropriate filter sets. Nuclear DNA can be simultaneously labeled at a final concentration of 0.1 /u.g/ml Hoechst 33342. If desired, samples can be fixed and stained simultaneously. A IO-/1I sample is mixed with 10 /x.1 of 7% paraformaldehyde containing 20 ju.g/ml of either of the lipophilic dyes. 

The fate of other nuclear components can concomitantly be analyzed using double-label immunofluorescence microscopy. Cells were fixed and air dried (see above), then incubated with antiguinea pig-specific secondary antibodies conjugated to FITC (10 min), and afterward washed in PBS (10 min). In a second step, the cover slips were incubated for 20 min with an antibody specific for a different nuclear protein (for example, a mouse monoclonal antibody against nucleolar protein fibrillarin). After washing in PBS (10 min), the cells were incubated with an appropriate secondary antibodies (in this case, antimouse-speciflc antibodies) conjugated to Texas Red (Dianova) for 10 min. For the visualization of chromatin, specimens were stained with the DNA-specific fluo-rochrome Hoechst 33258 (5 jug/ml in PBS) simultaneously with the secondary antibody. Finally, the cover slips were washed for 10 min in PBS, dehydrated in ethanol, and embedded in Mowiol (Hoechst, Frankfurt, Germany). 

Fig. 24 An indium bis(thiosemicarbazonato) complex (b) observed in HeLa cells using con-focal microscopy (a) by Pascu et al, indicating nuclear uptake by Hoechst staining (blue), where green signifies the complex and cyan denotes colocalisation. ...

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