All the primers used in this study were purchased from Thermo Fisher, gene blocks were purchased from IDT DNA, and restriction enzymes were purchased from New England BioLabs. The primary antibodies used in this study are the following: mouse α-V5 (clone SV5-Pk2, Bio-Rad); rabbit α-V5 (ICL, RV5-45A-Z); rat α-hemagglutinin (HA, clone 3F10, Sigma); mouse α-tubulin (clone B-5-1-2, Sigma); rabbit α-Histone H3 (ab1971, Abcam); mouse α-Centrin (CrCen clone 20H5, EMD Millipore); and rabbit α-PfGAP45 [a gift from Julian Rayner at the University of Cambridge (54 (link))]. Secondary antibodies and other reagents (Alexa Fluor 405 NHS-Ester, SYTOX Deep Red Nucleic Acid Stain, and Hoechst 33342 Solution) used for microscopy were purchased from Thermo Fisher.
Sytox deep red nucleic acid stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYTOX Deep Red Nucleic Acid Stain is a fluorescent dye used for nucleic acid detection. It exhibits increased fluorescence upon binding to DNA or RNA.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (2 mentions)
Hoechst 33342 solution, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 405 nhs ester, by Thermo Fisher Scientific (2 mentions)
Mouse α v5 clone sv5 pk2, by Bio-Rad (2 mentions)
Rabbit α histone h3 ab1971, by Abcam (2 mentions)
Mouse α centrin crcen clone 20h5, by Merck Group (2 mentions)
Mouse α tubulin clone b 5 1 2, by Merck Group (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Accutase, by Biowest (1 mentions)
4 protocols using sytox deep red nucleic acid stain
1
Antibody Panel for Microscopy Studies
2
Antibody Reagents for Microscopy Experiments
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All the primers used in this study were purchased from Thermo Fisher, gene blocks were purchased from IDT DNA, and restriction enzymes were purchased from New England Biolabs. The primary antibodies used in this study are the following: mouse α-V5 (clone SV5-Pk2, BioRad); rabbit α-V5 (ICL, RV5-45A-Z), rat α-hemagglutinin (HA, clone 3F10, Sigma); mouse α-tubulin (clone B-5-1-2, Sigma); rabbit α-Histone H3 (ab1971, Abcam); mouse α-Centrin (CrCen clone 20H5, EMD Millipore); and rabbit α-PfGAP45 (gift from Julian Rayner at the University of Cambridge [53 (link)]). Secondary antibodies and other reagents (Alexa Fluor 405 NHS-Ester, SYTOX Deep Red Nucleic acid stain, and Hoechst 33342 solution) used for microscopy were purchased from Thermo Fisher.
3
Quantification of Bacterial Colonization in Murine Colon
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Proximal colonic tissue samples were harvested and fixed in Carnoy’s fixative for a minimum of 72 h and then processed as previously described (54 (link)). Tissue sections (5 μm) were deparaffinized followed by hybridization for 4 h at 46°C in the presence of 20% formamide utilizing the following probes as reported earlier (71 (link)): pan-bacterial EUB338 (5′-GCTGCCTCCCGTAGGAGT -3′), non-EUB (5′-ACATCCTACGGGAGGC -3′) and species-specific ENTBAC (5′-CCTTGCGGTTGGCTTCAGAT -3′). Probes were labeled at the 5′ and 3′ ends with fluorescein isothiocyanate (FITC), Cy5, or Cy3 (BioTez, Berlin, Germany). After washing, cell nuclei were stained with Sytox Deep Red Nucleic Acid Stain (Invitrogen) and cover-slipped using ProLong Gold Antifade Reagent (Life Technologies). Samples were viewed and imaged in a blinded fashion on a Leica DMI4000 B inverted confocal microscope. FISH scores were obtained according to a previously reported scoring system with slight modifications (64 (link)). Briefly, epithelial attachment and epithelial invasion of microbes hybridizing with the Cy3-ENTBAC probe were assessed in 3 different regions of the proximal colon sample for each mouse. Bacterial quantities were enumerated as described in (64 (link)) and converted to a score from 1 to 4 for both parameters (i.e., a maximum score of 8).
4
Apoptosis and Necrosis Analysis of Compound 28 Treatment
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For the determination of apoptotic and necrotic cell death after treatment with compound 28, Annexin V-Sytox Deep Red staining was performed. Therefore, MDA-MB-231 and HS578T cells were seeded in 6-well plates. After 24 h, the cells were treated with different concentrations of compound 28 (10 nM, 100 nM, 250 nM, 500 nM, 1 µM, and 2 µM) for 24 h, 48 h, and 72 h at 37 °C and 5% CO2. For analysis of cell death, detached cells were collected in tubes and living cells were detached by accutase (Biowest, Nuaillé, France) and collected in the same tube. After several washing steps cells were resuspended in 1x annexin V binding puffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) and stained with 5 µL Annexin V-FITC (BioLegend, San Diego, CA, USA) and 1 µL 100 µM Sytox Deep Red Nucleic Acid Stain (Invitrogen, Thermo Fisher Scientific) for 15 min. Afterward, 400 µL 1x annexin V binding puffer were added to each tube. Gating was realized by the use of unstained, single Annexin V-FITC or single Sytox Deep Red Nucleic Acid-stained cells, respectively. For quantification of necrotic and apoptotic cells, 10,000 cells were analyzed by LSRFortessa™ flow cytometer (BD Biosciences, Heidelberg, Germany).
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