Bovine serum albumin (heat shock fraction, pH 7, ≥98%) and rhodamine 6G (Rho) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The glutaraldehyde solution (25% for synthesis) used for crosslinking was purchased from AppliChem GmbH (Darmstadt, German) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was from Tokyo Chemical Industry (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were from Gibco (New York, NY, USA). The Hoechst 33,342 solution and apoptosis kit were from Invitrogen (Waltham, MA, USA). The Cell Counting Kit-8 was from Enzo Life Sciences (New York, NY, USA). Ultrapure water (Merck, Darmstadt, Germany) was used for all experiments. All other reagents used were of analytical grade.
Hoechst 33342 solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Hoechst 33342 solution is a fluorescent dye that binds to DNA. It is commonly used in various laboratory applications, such as cell staining and flow cytometry, to visualize and analyze DNA content in cells.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Triton x 100, by Merck Group (6 mentions)
Propidium iodide, by Thermo Fisher Scientific (5 mentions)
Facsaria 2, by BD (3 mentions)
Lsm 700 confocal microscope, by Zeiss (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Prism 9, by GraphPad (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Goat anti rabbit igg fitc antibody, by Abcam (2 mentions)
L7543, by Merck Group (2 mentions)
Facscalibur, by BD (2 mentions)
Mitotracker red fm, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Sytox green nucleic acid staining, by Thermo Fisher Scientific (2 mentions)
Leica dmi 400b inverted microscope, by Leica camera (2 mentions)
Leica application suite advanced fluorescence, by Leica camera (2 mentions)
152 protocols using hoechst 33342 solution
1
Bovine Serum Albumin-Rhodamine 6G Conjugation
2
Imaging of Fluorescent Proteins in Arabidopsis
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All imaging of GFP, YFP, and RFP was performed with a laser-scanning confocal microscope (Zeiss LSM880 upright) and the areas below the hook and above the elongation zone of the hypocotyls of dark-grown seedlings were imaged. Samples were directly mounted on a glass slide in water containing with or without ACC. For imaging of Arabidopsis seedlings transformed with CTR1 constructs, the seedlings were grown on MS medium with or without 100 µM AgNO3 supplementation in the dark for 3 d. For ACC treatment, seedlings on MS without AgNO3 were treated with 200 µM ACC dissolved in water for 2 h. For ethylene treatment, seedlings were grown directly on MS medium in GC vials for 3 d. The GC vials were subsequently capped, injected with 10 ppm ethylene gas, and incubated for 2 h. For nuclear imaging, 3-d-old dark-grown seedlings were treated with 200 µM ACC for 2 h, incubated with Hoechst33342 solution (Invitrogen) for 30 min, and briefly washed before mounting on slides. For imaging light-grown seedlings, plants were grown on MS in constant light for 5 d. For imaging of fluorescence signals in protoplasts, transfected protoplasts were incubated with 200 µM ACC for 2 h in the dark and then examined. To image BiFC, leaf disks of infiltrated tobacco leaves with CTR1 and counterpart constructs (EIN2-CEND, EIN3, EBF1, EBF2, ENAP1, and CIP8) were mounted on glass slides in water.
3
Cell Cycle Stage Sorting Protocol
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Briefly, cells were stained with the following phycoerythrin-conjugated antibodies (Abs) CD34, Sca-1, CD45, c-kit, CD31, CD44 (BD Biosciences) and Ki-67 (eBiosciences) or Hoechst 33342 solution (Invitrogen). Flow cytometric analysis was performed by FACSCalibur or FACSAria II (BD Biosciences) and analyzed by Flowjo (Tree Star).
For sorting G0 and G1 cells, the transduced NIH3T3 cells were cultured in DMEM supplemented with 10% FBS for 5 hr after they were cultured in starvation medium. mVenus-p27K−(+)/mCherry-hCdt1(30/120)(+) NIH3T3 cells (G0) or mVenus-p27K− (−)/mCherry-hCdt1(30/120)(+) NIH3T3 cells (G1) were sorted using FACS AriaII.
For sorting G0 and G1 cells, the transduced NIH3T3 cells were cultured in DMEM supplemented with 10% FBS for 5 hr after they were cultured in starvation medium. mVenus-p27K−(+)/mCherry-hCdt1(30/120)(+) NIH3T3 cells (G0) or mVenus-p27K− (−)/mCherry-hCdt1(30/120)(+) NIH3T3 cells (G1) were sorted using FACS AriaII.
4
Immunophenotyping of Ovarian Cancer-Associated Fibroblasts
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Immunocytochemistry of cytokeratin 19 (CK19), α-smooth muscle actin (α-SMA), vimentin (VIM) and fibroblast activation protein (FAP) was performed to verify the purity of OVCAFs and OVNFs. Briefly, cells on sterile glass coverslips were fixed in ice-cold methanol and then incubated overnight at 4°C in a humid chamber with the indicated primary antibodies as follow: 1:200 mouse anti-human CK-19 antibody (sc-6278, Santa Cruz Biotechnology Inc.), 1:500 anti-α-SMA antibody (A5228, Abcam), 1:500 anti-VIM antibody (sc-6260, Santa Cruz Biotechnology Inc.), and 1:500 rabbit anti-human FAP (ab53066, Abcam). After washing out the excess primary antibody, the coverslip was incubated for 3 h at room temperature with the appropriate secondary fluorescent antibody including goat anti-mouse IgG-Cy3 antibody (1:2,000, #115-166-071, Jackson ImmunoResearch Laboratories Inc.) or the goat anti-rabbit IgG-FITC antibody (1:2,000, ab6717, Abcam). Hoechst 33342 solution (Invitrogen; Thermo Fisher Scientific, Inc.) was added to stain the nucleus. Fluorescence was captured using an Inverted microscope model IX71 (Olympus Corporation).
5
Assessing Cell Viability and Morphology
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Both cell morphology and cell viability were assessed after 1, 4, and 7 days of culture in AM. For morphological observation, the cell-laden scaffolds were fixed in 4% paraformaldehyde (PFA) aqueous solution (Fisher Scientific, Hampton, NH) for 2 h and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). Alexa Fluor 488 phalloidin (Invitrogen, Carlsbad, CA) was used to label cytoskeletal actin filaments, and Hoechst 33342 solution (Invitrogen, Carlsbad, CA) was utilized as nuclear counterstain. To assess cell viability, the LIVE/DEAD cell viability assay (Life Technologies, Carlsbad, CA) was used. Briefly, constructs were transferred into maintenance medium (phenol red-free DMEM (Gibco) containing 10% (v/v) FBS), containing calcein AM and ethidium homodimer-1, and then incubated at 37°C for 30 min. Images were acquired using an Olympus Fluoview 1000 confocal microscope (Center Valley, PA). Z stacks were acquired at optimal intervals (2 μm or 4 μm steps, 100–150 μm stack) as suggested by the software. NIH ImageJ software was utilized to analyze all the confocal stacks.
6
Fordin-Induced Cell Morphology Changes
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The selected cells were seeded into 12-well plates containing sterilized coverslips. Following treatment with fordin (IC20, IC50, or IC70) for 24 h, the cells were fixed with 4% paraformaldehyde, and stained with 4 µg/ml of Hoechst 33342 solution (Invitrogen; Thermo Fisher Scientific, Inc.). The coverslips with cells were inverted onto slides. Six random images were captured with the inverted fluorescent microscope.
7
Mitochondrial and Nuclear Labeling in hASM Cells
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hASM cells were plated at a density of ~15,000 cells/well into 8-well Ibidi μ-slide plates (Ibidi GmbH, Gewerbehof Gräfelfing, Germany) and incubated to allow for cell adherence and subsequently serum deprived. The cells were loaded with 200 nM MitoTracker® Red FM (Invitrogen, Carlsbad, CA, USA; excitation/emission wavelength: 581/644 nm) or 200 nM MitoTracker® Green FM (Invitrogen, Carlsbad, CA, USA; excitation/emission wavelength: 490/516 nm) and 1 µg/mL Hoechst 33342 Solution (Invitrogen, Carlsbad, CA, USA; excitation/emission wavelength: 361/486 nm), to label the nucleus, in serum-free media pre-warmed to 37 °C, for 15 min followed by extensive washing with 1X Hanks’ Balanced Salt Solution (HBSS).
8
Cellular Epifluorescence Imaging Protocol
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The cellular preparation for epifluorescence was made as follows. After 48 h of incubation in 48-well plates, the supernatant was removed. Then, each well was washed with 500 μL PBS, and 100 μL of MitoTracker Green 250 nM (Invitrogen, USA) was added. The plates were incubated at 37°C for 30 min. After washing with 500 μL PBS, 100 μL of CellMask Orange 1X (Molecular Probes) was added. The preparations were incubated at 37°C for 5 min. After washing with 500 μL PBS, 100 μL of 0.1 μg/mL Hoechst 33342 solution (Invitrogen) was added. The plates were incubated at 37°C for 10 min; later, digital images were taken using an EVOS FL Imaging System (Life Technologies, USA). Micrographs were analyzed with ImageJ Software v 1.50i (Wayne Rasband, National Institutes of Health, USA) to obtain fluorescence intensity due to MitoTracker Green per cell line.
9
Mitotic Cell Imaging in CO2-Independent Medium
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The cells were transferred to PLL‐g‐PEG coated fluorodishes (FD35‐100) with CO2‐independent culture medium (described previously). Cells were stained with Hoechst 33342 solution (PN:62249, Invitrogen) in order to distinguish between mitotic and interphase cells. During imaging they were maintained at 37 °C using ibidi heating stage. Images were recorded using a Zeiss LSM700 confocal microscope of the CMCB light microscopy facility, incorporating a Zeiss C‐Apochromat 40×/1.2 water objective. Images were taken at the equatorial diameter of each cell exhibiting the largest cross‐sectional area.
10
Neutrophil Extracellular Traps in Cancer
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The second plate with coculture of neutrophils and SCC cells (105 PMNs
vs. 0.5 × 105 SCC in the well) or PMNs stimulated with the
supernatant obtained from SCC cell culture (100 µL) was incubated in the BD
Pathway 855 system. Cells were suspended in an RPMI-1640, FBS Good (<5%) and
Penicillin-Streptomycin (1%) medium and incubated at 37°C with 5% CO2flow in a 96-well Black/Clear Tissue Culture Treated Imaging Plate with a Flat
Bottom and a Lid (BD Falcon). Cell growth was recorded live using a microscopic
system. A Hoechst 33342 solution (Invitrogen) was used for DNA staining while
myeloperoxidase was visualized through the use of fluorescein-labeled monoclonal
antibodies (Clone 8E6, Molecular Probes).
Determination of the percentage of NETs-forming cells occurred on the basis of
same-time photos taken by a microscopic system. NETs-forming neutrophils and
normal cells were counted in the field of view/photos (9 in each well). On this
basis, the average percentage of NETs forming neutrophils was determined.
vs. 0.5 × 105 SCC in the well) or PMNs stimulated with the
supernatant obtained from SCC cell culture (100 µL) was incubated in the BD
Pathway 855 system. Cells were suspended in an RPMI-1640, FBS Good (<5%) and
Penicillin-Streptomycin (1%) medium and incubated at 37°C with 5% CO2flow in a 96-well Black/Clear Tissue Culture Treated Imaging Plate with a Flat
Bottom and a Lid (BD Falcon). Cell growth was recorded live using a microscopic
system. A Hoechst 33342 solution (Invitrogen) was used for DNA staining while
myeloperoxidase was visualized through the use of fluorescein-labeled monoclonal
antibodies (Clone 8E6, Molecular Probes).
Determination of the percentage of NETs-forming cells occurred on the basis of
same-time photos taken by a microscopic system. NETs-forming neutrophils and
normal cells were counted in the field of view/photos (9 in each well). On this
basis, the average percentage of NETs forming neutrophils was determined.
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