Adjudin (ADD) was synthesized at S.B.M. Srl (Rome, Italy) with a purity of >98% as described earlier.30 (link) Doxorubicin hydrochloride (DOX·HCl) was purchased from Huafeng United Technology Co., Ltd (Beijing, China). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). DMSO for cellular experiments was purchased from Sigma-Aldrich (St Louis, MO, USA). Lysotracker Green DND-26 was purchased from Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Hoechst 33258 were purchased from Bio-sharp (Seoul, South Korea). The ATP Bioluminescence Assay Kit and the BCA Protein Assay Kit were from Beyotime Institute of Biotechnology (Shanghai, China). RPMI-1640 medium, penicillin–streptomycin, fetal bovine serum (FBS) and trypsin without EDTA were purchased from Hyclone (USA). All other non-mentioned reagents were obtained from Aladdin (Shanghai, China).
Hoechst 33 258
Manufactured by Biosharp
Sourced in China
Hoechst 33,258 is a fluorescent dye used for nucleic acid detection. It binds to the minor groove of DNA, resulting in a fluorescent signal that can be detected and measured.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n methoxy polyethylene glycol 2000 dspe peg2000, by Avanti Polar Lipids (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Atp bioluminescence assay kit, by Beyotime (1 mentions)
3 4 5 dimethyl thiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Biosharp (1 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Blyscan sgag assay, by Biocolor (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Hoechst 33258, by Beyotime (1 mentions)
Ficoll paque, by Merck Group (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
5 protocols using hoechst 33 258
1
Preparation and Characterization of Adjudin-Doxorubicin Nanoparticles
2
Quantitative Analysis of GAG and DNA
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At the specified time points, the cells were washed with PBS three times and then were frozen in liquid nitrogen and lyophilized for 12 h. The remaining cells were collected and digested with the GAG digestion solution (papain 0.0156 g, NaOH 0.2 g, EDTA 0.0731 g, sodium dihydrogen phosphate 0.39 g, and cysteine 0.0304 g in 25 ml ultrapure water) for 16 h at 60°C. The GAG content was measured using the Blyscan sGAG Assay (Biocolor, United Kingdom), and the standard curve was generated by chondroitin sulfate standard using the kit. The DNA content was quantified using Hoechst 33,258 (Biosharp, China). Calf thymus DNA (Sigma) was used to generate a standard curve (Farndale et al., 1986 (link)).
3
Hoechst 33,258 Assay for BLCA Cell Apoptosis
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The induction of BLCA cell apoptosis with different concentrations of HE was assessed by analyzing Hoechst 33,258 (Beyotime, Shanghai) staining of apoptotic cell nuclei. The cells were cultured in a 24-well plate. Hoechst 33,258 solution was added to the cells for 48 h after HE intervention, and the cells were fixed with 4% paraformaldehyde (Biosharp, Guangzhou). The samples were washed twice with PBS and then observed under white light and blue light, respectively, to observe the morphology of the cell nucleus.
4
Selective Tumor Cell Identification
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We co-cultured MDA-MB-231 and GFP-MCF-10A cells in confocal dishes with microscope slides to investigate whether the nanoprobe could selectively recognize tumor cells and differentiate these from normal human epithelial cells. Normal GFP-MCF-10A epithelial cells were labeled with a green fluorescence marker using transfected GFP-actin plasmid DNA in 3.5 cm dishes plated at a density of 1 × 104 cells/dish. After MCF-10A cells had adhered to the dishes, MDA-MB-231 cells were added to the same dishes at the same density, and all cells were labeled with a blue fluorescence marker by Hoechst 33,258 (BioSharp, BL804A, China). The cells were then incubated with nanoprobe (5 μM) at 37 °C for 6 h [28 (link)]. The images from multiple dishes were obtained by the confocal microscopy (Zessis, LSM800, Germany).
5
Evaluating PROTAC-Induced T Cell Cytotoxicity
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Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor in an ethylenediamine tetraacetic acid anticoagulant tube by Ficoll-Paque (Sigma-Aldrich) via density gradient separation. CD3 T cells were isolated from PBMCs using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies). C33A cells were then seeded and cocultured with the T cells at a 1:2 ratio (2.5 × 104 in 50 μl/well and 5 × 104 in 100 μl/well, respectively) in a 96-well plate at 37°C under 5% CO2. Then, SP-PROTAC and BMS-8 were added as described and maintained for 4 h. The 96-well plates were washed with PBS and stained with Hoechst 33258 (Biosharp) staining solution. The cells were washed twice and observed by fluorescence microscopy using appropriate bandpass filters.
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