Bone marrow cells were analyzed for cell cycle status as described [2 (link), 6 ]. Cell cycle phases were determined by staining cells with Hoechst 33343 (Sigma Aldrich) and APC-conjugated anti-Ki-67 (SolA15,eBioscience).
Hoechst 33343
Manufactured by Merck Group
Sourced in United States
Hoechst 33343 is a fluorescent dye used for staining nuclei in living and fixed cells. It binds to DNA and emits blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Apc conjugated anti ki 67 sola15, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti s100, by Agilent Technologies (1 mentions)
Rabbit anti sox10, by Abcam (1 mentions)
Rabbit anti p75ntr, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Rabbit anti sox2, by Merck Group (1 mentions)
Rabbit anti pax6, by BioLegend (1 mentions)
Mouse anti ki 67, by Abcam (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Mouse anti βiii tubulin, by Fortrea (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Ix81 fluorescence microscope, by Olympus (1 mentions)
7 protocols using hoechst 33343
1
Cell Cycle Analysis of Bone Marrow
2
Cell Cycle Analysis of Bone Marrow
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Bone marrow cells were extracted in the morning and analyzed for cell cycle status as described [39 (link)]. Cell cycle phases were determined by staining cells with Hoechst 33343 (Sigma Aldrich) and APC-conjugated anti-Ki-67 (SolA15,eBioscience). For these experiments, FITC-conjugated anti-mouse lineage marker antibodies were used.
3
Immunostaining of Stem Cells and Progenitor Cells
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hE-SCs/P0, hE-SCs/P2,and hE-FLCs/P2 were trypsinized and replated on cover slides for immunostaining. The cells were fixed with 4% paraformaldehyde (Sigma) in PBS for 15 min. The fixed cells were washed three times with PBS for 5 min each, and then, the cells were blocked with 10% bovine serum albumin (Sigma, USA) in PBS for 30 min at room temperature, followed by three washes with PBS. The cells were then incubated with rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Abcam, Cambridge, UK), rabbit anti-P75NTR (1:500 diluted in PBS; Abcam, Cambridge, UK), rabbit anti-S100 (1:500; Dako, Glostrup, Denmark), rabbit anti-Sox10 (1:500 diluted in PBS; Abcam, Cambridge, UK), and rabbit anti-Krox10 (1:500 diluted in PBS; Abcam, Cambridge, UK) overnight at 4 °C. After being washed with PBS, the slides were treated with Alexa Fluor 488 goat anti-rabbit IgG (1:500; Invitrogen, USA) at 37 °C for 60 min. After they were washed with PBS, the cells were incubated with 1 M Hoechst 33343 (Sigma, USA) for 10 s. Labeled cells were examined with fluorescence microscopy (Olympus, Japan), and the images were recorded and processed with Image-Pro Plus (Media Cybernetics, USA).
4
Bone Marrow Cell Cycle Analysis
5
Immunofluorescence Staining of Cultured Cells
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Cultured cells were fixed in 4% paraformaldehyde for 10 min, then blocked with 10% normal goat serum (NGS) diluted in PBS with 0.3% Triton X-100. Primary antibodies were diluted in 10% NGS in PBS with 0.3% Triton X-100 and incubated in a humid chamber at 4 °C overnight. Secondary antibodies were diluted in PBST and incubated for 1 h at room temperature. Hoechst 33258 was diluted in PBS. The culture was washed three times for 5 min with PBS between each step. The primary antibodies used were mouse anti-βIII-tubulin (1:1000; Covance, Princeton, NJ, USA), rabbit anti-Pax-6 (1:2000; BioLegend, San Diego, CA, USA), rabbit anti-Sox2 (1:100; Millipore, Burlington, MA, USA), mouse anti-Ki67 (1:400; Abcam, Cambridge, UK), and rabbit anti-Cleaved Caspase-3 (CC3) (1:400; Cell Signalling Tech., Danvers, MA, USA). The secondary antibodies used were Alexa Fluor 555- and Alexa Fluor 488-conjugated goat antibodies (1:500; Life Technologies, Carlsbad, CA, USA). Nuclear staining was performed with Hoechst 33343 (1:1000; Sigma, St. Louis, MO, USA). After rinsing with PBS, the coverslips were mounted in a Lab Vision PermaFluor Aqueous Mounting Medium (Thermo Fisher, Waltham, MA, USA). All experiments were repeated at least three times.
6
Immunostaining of p75NTR in Cultured Cells
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Cells cultured on cover slips were fixed with 4% paraformaldehyde for 20 minutes, washed three times with PBS, and then blocked with 10% goat serum (Sigma) in PBS for 30 minutes at 37°C. Samples were treated with rabbit polyclonal anti-p75NTR antibody (Abcam, Cambridge, UK) diluted 1:500 in PBS at 37°C for 1 hour. As a control, samples were incubated without primary antibody. After three washes with PBS, samples were treated with Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) diluted 1:1,000 in PBS for 30 minutes at 37°C. Cell nuclei were counterstained with 1 μM Hoechst 33343 (Sigma) for 10 seconds. After a final wash in PBS, samples were mounted with mounting medium (Dako) and visualized under a fluorescence microscope (Olympus). Images were captured and processed with Image-Pro Plus software (Media Cybernetics). The cell morphologies were observed and classified, and the SC purity was calculated based on p75NTR immunostaining.
7
Apoptosis assessment using Hoechst-PI staining
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After treatment with GSK343 +/-TRAIL for 24 hours apoptosis induction was assessed by Hoechst 33343 and propidium Iodide (PI) stain (Sigma-Aldrich, Dorset, England).
Cells were incubated with 10µg/ml Hoechst 33342 and 10µg/ml PI at 37°C for 30 minutes examined using IX81 Olympus fluorescence microscope, using Cell F software for image capture. Images were captured using a UC30 colour camera and triple filter for DAPI/FITC/TRITC. Images containing at least 100 cells were obtained from duplicate wells in three technical repeats and apoptosis calculated by manual counting of cells with characteristic condensed or pyknotic nuclei. PI was used to exclude necrosis.
Cells were incubated with 10µg/ml Hoechst 33342 and 10µg/ml PI at 37°C for 30 minutes examined using IX81 Olympus fluorescence microscope, using Cell F software for image capture. Images were captured using a UC30 colour camera and triple filter for DAPI/FITC/TRITC. Images containing at least 100 cells were obtained from duplicate wells in three technical repeats and apoptosis calculated by manual counting of cells with characteristic condensed or pyknotic nuclei. PI was used to exclude necrosis.
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