When staining for surface glycoproteins, 50 μg/ml Alexa Fluor® 555-conjugated WGA (wheat-germ agglutinin; Invitrogen) was applied to the cells on ice for 5 min in DMEM (without Phenol Red or sodium pyruvate) supplemented with 25 mM Hepes (Invitrogen) and 10% FBS, rinsed twice in the same solution and then fixed as above. Staining for plasma membrane PtdIns(4,5)P2 or endosomal PtdIns3P were performed as described in the preceding two sections. For staining of the endoplasmic reticulum, after post-fixation and rinsing in PBS/NH4Cl, cells were incubated for 1 min with DiOC6 (3,3′-dihexyloxacarbocyanine iodide; Invitrogen) in PBS, rinsed once with distilled water and mounted in ProLong Gold with 1 μg/ml DAPI as above. A similar protocol was used to stain cells with 500 μM DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; Invitrogen). For Golgi staining, the slides were chilled on ice for 2 min following the post-fixation and rinsing. They were incubated with a 5 μM solution of NBD-C6-ceramide [N-(ε-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D -erythro-sphingosine] complexed to BSA (Invitrogen) in PBS for 30 min on ice. Unbound ceramide was removed with two rinses in ice-cold PBS, and extra-Golgi ceramide was extracted by four washes with 3% (w/v) defatted-BSA (Sigma–Aldrich) in PBS over 1 h at room temperature. Finally, slides were stained for 5 min in 1 μg/ml DAPI in PBS, rinsed once in distilled water and mounted in Fluoromount-G (Southern Biotech). Note that we found retention of this latter dye was poor in glycerol-based mountants, hence the use of aqueous Fluoromount-G. Furthermore, slides stained with NBD-C6-ceramide were imaged promptly after staining, since we noticed progressive loss of the stain from the Golgi over a time course of hours.
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3,3'-dihexaoxycarbocyanine iodide
3,3'-dihexaoxycarbocyanine iodide
3,3'-dihexaoxycarbocyanine iodide is a fluorescent dye commonly used in biological and medical research.
It is a cyanine dye with a hexaoxycarbocyanine structure, making it useful for staining and visualizing cellular structures and processes.
This dye has been widely employed in applications such as flow cytometry, fluorescence microscopy, and cellular imaging to label and track various biological entities.
Optimizing protocols for the use of 3,3'-dihexaoxycarbocyanine iodide can be challening, but with the AI-powered tools of PubCompare.ai, researchers can quickly identify the most efficient and reliable protocols from the literature, pre-prints, and patents.
This AI-assited optimization helps ensure reproducible and effective results in 3,3'-dihexaoxycarbocyanine iodide-based studies.
It is a cyanine dye with a hexaoxycarbocyanine structure, making it useful for staining and visualizing cellular structures and processes.
This dye has been widely employed in applications such as flow cytometry, fluorescence microscopy, and cellular imaging to label and track various biological entities.
Optimizing protocols for the use of 3,3'-dihexaoxycarbocyanine iodide can be challening, but with the AI-powered tools of PubCompare.ai, researchers can quickly identify the most efficient and reliable protocols from the literature, pre-prints, and patents.
This AI-assited optimization helps ensure reproducible and effective results in 3,3'-dihexaoxycarbocyanine iodide-based studies.
Most cited protocols related to «3,3'-dihexaoxycarbocyanine iodide»
3,3'-dihexaoxycarbocyanine iodide
Alexa Fluor 555
Cells
Ceramides
Cold Temperature
DAPI
Endoplasmic Reticulum
Endosomes
erythro-(2R,3S)-sphingosine
Glycerin
Gold
Golgi Apparatus
HEPES
Iodides
Membrane Glycoproteins
N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine
perchlorate
phosphatidylinositol 3-phosphate
Phosphatidylinositols
Plasma Membrane
Pyruvate
Retention (Psychology)
Sodium
Stains
Wheat Germ Agglutinins
Animal work was performed according the Animals (Scientific Procedures) Act, UK. Dicerlox/lox mice were crossed with LckCre transgenic mice (17 (link)) to generate lckCre DicerΔ/Δ mice. Thymocytes were stained, analyzed, and sorted by flow cytometry as described previously (24 (link)). Where indicated, thymocytes were incubated with 40 nM DiOC6 (Molecular Probes) for 10 min at 37°C as described previously (20 (link)). To down-regulate Tdt expression, DP thymocytes were cultured with 7.5 ng/ml PMA (Sigma-Aldrich) and 180 ng/ml ionomycin (Sigma-Aldrich) as described previously (28 (link)).
3,3'-dihexaoxycarbocyanine iodide
Animals
Flow Cytometry
Ionomycin
Lanugo
Mice, Laboratory
Mice, Transgenic
Molecular Probes
Thymocyte
3,3'-dihexaoxycarbocyanine iodide
dichlorofluorescin
dihydroethidium
dihydrorhodamine 123
Esters
Flow Cytometry
Fluorescent Probes
Iodides
Mitochondria
Molecular Probes
N(10)-nonylacridine orange
Reactive Nitrogen Species
tetramethylrhodamine
Trees
Quinacrine (Sigma) staining was performed as previously described39 (link). Briefly, 2 × 106 log-phase or purified aged cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 200 μM quinacrine. Cells were incubated for 10 min at 30°C and then 5 min on ice. Cells were pelleted and washed twice with ice cold 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging. Prior to imaging, cells were kept on ice and all images were obtained within 30 min of staining.
For staining with 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate11 (link) (CDCFDA) (Invitrogen) or 2′,7′-bis (carboxyethyl)-5(6)-carboxyfluorescein40 (link) (BCECF-AM) (Invitrogen), 2 × 106 log-phase or purified aged cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 10 μM CDCFDA or 50 μM BCECF. Cells were incubated for 30 min at 30°C and then washed twice with RT 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging.
3,3′-dihexyloxacarbocyanine iodide (DiOC6) (Invitrogen) staining was carried out according to manufacturer’s instructions. Briefly, 2 × 106 log-phase or purified aged cells were washed once in 10 mM HEPES, pH 7.6 + 5% glucose and resuspended in 1 ml of the same buffer containing 175 nM DiOC6. Cells were incubated for 15 min at RT and then washed twice with 10 mM HEPES, pH 7.6 + 5% glucose. Cells were resuspended in 10 mM HEPES, pH 7.6 + 5% glucose for imaging. Pho8-SEP imaging was also carried out in DiOC6 imaging buffer after incubation of cells for 20 minutes in the buffer. In all live cell experiments, calcofluor (Sigma) staining of bud scars for age determination was carried out by including 5 μg/ml calcofluor in the first post-staining wash step prior to imaging.
For fluorescence microscopy analysis, cells were visualized under 60X oil magnification using a Nikon Eclipse E800 with the appropriate filter set: UV-2E/C DAPI for calcofluor; FITC-HYQ for GFP, SEP, quinacrine, CDCFDA, BCECF, and DiOC6; and G-2E/C TRITC for mCherry. Images were acquired with a CoolSNAP HQ2 CCD camera (Photometrics) and quantified and processed using Metamorph version 7.1.1.0 imaging software. Cells that exhibited at least four-fold reduction in mean fluorescence intensity, compared to young, wild type cells, were scored as “reduced” in all figures.
For staining with 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate11 (link) (CDCFDA) (Invitrogen) or 2′,7′-bis (carboxyethyl)-5(6)-carboxyfluorescein40 (link) (BCECF-AM) (Invitrogen), 2 × 106 log-phase or purified aged cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 10 μM CDCFDA or 50 μM BCECF. Cells were incubated for 30 min at 30°C and then washed twice with RT 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging.
3,3′-dihexyloxacarbocyanine iodide (DiOC6) (Invitrogen) staining was carried out according to manufacturer’s instructions. Briefly, 2 × 106 log-phase or purified aged cells were washed once in 10 mM HEPES, pH 7.6 + 5% glucose and resuspended in 1 ml of the same buffer containing 175 nM DiOC6. Cells were incubated for 15 min at RT and then washed twice with 10 mM HEPES, pH 7.6 + 5% glucose. Cells were resuspended in 10 mM HEPES, pH 7.6 + 5% glucose for imaging. Pho8-SEP imaging was also carried out in DiOC6 imaging buffer after incubation of cells for 20 minutes in the buffer. In all live cell experiments, calcofluor (Sigma) staining of bud scars for age determination was carried out by including 5 μg/ml calcofluor in the first post-staining wash step prior to imaging.
For fluorescence microscopy analysis, cells were visualized under 60X oil magnification using a Nikon Eclipse E800 with the appropriate filter set: UV-2E/C DAPI for calcofluor; FITC-HYQ for GFP, SEP, quinacrine, CDCFDA, BCECF, and DiOC6; and G-2E/C TRITC for mCherry. Images were acquired with a CoolSNAP HQ2 CCD camera (Photometrics) and quantified and processed using Metamorph version 7.1.1.0 imaging software. Cells that exhibited at least four-fold reduction in mean fluorescence intensity, compared to young, wild type cells, were scored as “reduced” in all figures.
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester
3,3'-dihexaoxycarbocyanine iodide
Buffers
Cells
Cicatrix
Common Cold
DAPI
Fluorescein-5-isothiocyanate
Fluorescence
Glucose
HEPES
Iodides
Microscopy, Fluorescence
Quinacrine
tetramethylrhodamine isothiocyanate
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester
3,3'-dihexaoxycarbocyanine iodide
Buffers
Cells
Cicatrix
Common Cold
DAPI
Fluorescein-5-isothiocyanate
Fluorescence
Glucose
HEPES
Iodides
Microscopy, Fluorescence
Quinacrine
tetramethylrhodamine isothiocyanate
Most recents protocols related to «3,3'-dihexaoxycarbocyanine iodide»
Bovine Collagen type I, 8 wt.% aqueous solution (Coll, Collado s.r.o., Brno, Czech Republic), chitosan from shrimp shells, 70% DDA, low viscosity (Chit, Sigma-Aldrich, Darmstadt, Germany), calcium salt of oxidized cellulose–degree of oxidation 16–24% and Mn = 350 kg/mol (CaOC, Synthesia, Pardubice, Czech Republic), acetic acid (99%, Penta s.r.o, Chrudim, Czech Republic), poly(ε-caprolactone) (PCL, 80 kg/mol), gelatin (Gel, Type B, Bioreagent, powder from bovine skin), N-(3-Dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 98% dopamine hydrochloride, tris (hydroxymethyl) aminomethane hydrochloride, ethanol p.a. 99.8%, sodium phosphate dibasic for molecular biology (≥ 98,5%), sodium chloride, calcium chloride, sodium phosphate dibasic dodecahydrate (Na2HPO4 ·12H2O), potassium dihydrogen phosphate (KH2PO4), potassium chloride (KCl), collagenase from Clostridium histolyticum, lysozyme human, the murine fibroblast cell lines 3T3-A31, Dulbecco's modified eagle medium DMEM (D6429), fetal bovine serum FBS (F7524), 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), propidium iodide (P4864), (all from Sigma Aldrich, Darmstadt, Germany), penicillin/streptomycin (15140–122) and DiOC6(3) (D273), (Life Technologies, Eugene, OR, USA), octenidine solution (Octenisept®, Schülke, Germany), Butomidor® inj. (butorphanol tartrate, Vétoquinol, Czech republic), Domitor®, Medetomidine, Orion corporation, Finland) Propofol® (Propofolum 1%, Fresenius Kabi Deutschland, Bad Homburg, Germany), Metacam® (meloxicam, Boehringer Ingelheim Vetmedica, Ingelheim/Rhein, Germany), Enroxil® (Enrofloxacin, Krka, Novo mesto, Slovenia) Betadine®, (2.5% solution of povidone iodine, EGIS Pharmaceuticals PLC, Budapest, Hungary) were used as received without further purification.
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2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester
3,3'-dihexaoxycarbocyanine iodide
Acetic Acid
Betadine
Bos taurus
Butorphanol Tartrate
Calcium, Dietary
Calcium chloride
caprolactone
carboxyfluorescein
Cell Lines
Chitosan
chloropentaammineosmium(III) chloride
Collagenase, Clostridium histolyticum
Collagen Type I
Eagle
Enrofloxacin
Esters
Ethanol
Fibroblasts
Gelatins
Homo sapiens
Hydrochloride, Dopamine
Medetomidine
Meloxicam
methylamine hydrochloride
Muramidase
Mus
N-hydroxysuccinimide
octenidine
Octenisept
Oxidized Cellulose
Penicillins
Pharmaceutical Preparations
Poly A
potassium phosphate, monobasic
Povidone Iodine
Powder
Propidium Iodide
Propofol
rhein
Skin
Sodium Chloride
sodium phosphate
Streptomycin
Tetranitrate, Pentaerythritol
Tromethamine
Viscosity
Murine fibroblast cell lines 3T3-A31 were cultured in culture medium containing DMEM (high glucose, D6429, Sigma-Aldrich, St. Louis, MO, USA), 10% FBS, and 1% penicillin/streptomycin. 70,000 cells/scaffold (with a diameter of 10 mm and a height of 4–5 mm) were seeded for a period of 14 days.
Metabolic activity was determined by the CellTiter 96® Aqueous One Solution Cell Proliferation (MTS) metabolic assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega corp., Madison, WI, USA), where the MTS tetrazolium compound was added directly to the cell culture medium in a 1:5 ratio. Metabolically active cells reduced the MTS reagent and generated a colored formazan dye that is soluble in cell culture medium. Formazan dye was quantified by measuring the absorbance at 490 nm, reference 690 nm using Tecan Infinite M200 Pro. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
The Quant-iT™ dsDNA Assay Reagent (Invitrogen) assay determined cell proliferation, as it quantified the amount of double-stranded DNA. The assay contains a fluorescent dye activated once it is bound to dsDNA. Fluorescence was measured at λex = 485 nm and λem = 523 nm. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
Cell viability was assessed by live-dead staining of three samples. 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF, Sigma-Aldrich, Saint Luis, MO, USA) was used to visualize the membranes of living cells and propidium iodide to visualize the nuclei of dead cells. Samples were observed using a Zeiss LSM 880 Airyscan confocal microscope. Excitation/emission was set as follows: BCECF λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm.
The cell distribution on the scaffold and the morphology were observed using 3,3'-Dihexyloxacarbocyanine Iodide (Invitrogen™) DiOC6(3)/propidium iodide (ThermoFisher Scientific™) staining. Excitation/emission was set as follows: DiOC6(3) λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm. The signal from the nuclei was further used to determine the depth of penetration of the cell into the scaffold.
The statistical significance was performed using one-way analysis of variance (ANOVA) in Sigma Stat software 3.5 (Systat Software, California, USA).
Metabolic activity was determined by the CellTiter 96® Aqueous One Solution Cell Proliferation (MTS) metabolic assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega corp., Madison, WI, USA), where the MTS tetrazolium compound was added directly to the cell culture medium in a 1:5 ratio. Metabolically active cells reduced the MTS reagent and generated a colored formazan dye that is soluble in cell culture medium. Formazan dye was quantified by measuring the absorbance at 490 nm, reference 690 nm using Tecan Infinite M200 Pro. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
The Quant-iT™ dsDNA Assay Reagent (Invitrogen) assay determined cell proliferation, as it quantified the amount of double-stranded DNA. The assay contains a fluorescent dye activated once it is bound to dsDNA. Fluorescence was measured at λex = 485 nm and λem = 523 nm. The samples were carried out in biological quadruplicates; the results are shown as mean ± standard deviation.
Cell viability was assessed by live-dead staining of three samples. 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF, Sigma-Aldrich, Saint Luis, MO, USA) was used to visualize the membranes of living cells and propidium iodide to visualize the nuclei of dead cells. Samples were observed using a Zeiss LSM 880 Airyscan confocal microscope. Excitation/emission was set as follows: BCECF λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm.
The cell distribution on the scaffold and the morphology were observed using 3,3'-Dihexyloxacarbocyanine Iodide (Invitrogen™) DiOC6(3)/propidium iodide (ThermoFisher Scientific™) staining. Excitation/emission was set as follows: DiOC6(3) λex = 488 nm/λem = 505–545 nm, PI λex = 560 nm/λem ˃ 575 nm. The signal from the nuclei was further used to determine the depth of penetration of the cell into the scaffold.
The statistical significance was performed using one-way analysis of variance (ANOVA) in Sigma Stat software 3.5 (Systat Software, California, USA).
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2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
3,3'-dihexaoxycarbocyanine iodide
Biological Assay
Biopharmaceuticals
carboxyfluorescein
Cell Culture Techniques
Cell Lines
Cell Nucleus
Cell Proliferation
Cell Survival
Culture Media
DNA, Double-Stranded
Esters
Fibroblasts
Fluorescence
Fluorescent Dyes
Formazans
Glucose
Iodides
M-200
Microscopy, Confocal
Mus
Penicillins
Promega
Propidium Iodide
Streptomycin
Tetrazolium Salts
Microalgae cell counting and registration of morphological and biochemical changes were carried out using flow cytometer CytoFLEX (Beckman Coulter, Indianapolis, IN, USA) with the software package CytExpert v.2.0. The changes in microalgae cells after the exposure to welding suspensions were evaluated using specific fluorescent dyes. All the measurements with each fluorescent dye were performed separately after 96 h and 7 days of exposure. Each sample was measured at a flow rate of 100 μL/min for 30 s. The emission channels were selected according to the data provided by the manufacturer (Molecular Probes, Eugene, OR, USA). The blue laser (488 nm) of the CytoFLEX flow cytometer was chosen as a source of excitation light. The data of flow cytometry measurements were expressed as mean fluorescence intensity (MFI). The endpoints of toxicity used in this work and the parameters of their registration are listed in Table 1 .
The number of alive microalgae cells in each measurement was determined using FSC/SSC dot cytogram (forward scattering to side scattering ratio) which allowed for the separation of the population of events with the sizes similar to the expected size of microalgae cells. Then, nonalgal events were excluded from the separated population by the absence of chlorophyll a fluorescence in the emission filter FL3 (690 nm), where all the algal cells had high MFI. Dead cells were excluded from the counting by them staining with propidium iodide (PI) according to the standard bioassay protocol [33 (link)], where dead cells obtained high MFI in the emission filter FL1 (610 nm).
The level of reactive oxygen species (ROS) generation in microalgae cells was assessed using non-fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), which activates in the presence of ROS [34 (link)]. The membrane potential of microalgae cells was assessed by a lipophilic, positively charged fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6), which is capable of binding to membranes (mitochondria and endoplasmic reticulum) and other hydrophobic negatively charged cell structures [35 (link)]. Both ROS generation and membrane potential were registered as MFI in the emission filter FL2 (525 nm) compared to the MFI of the control group in the same emission channel. The evaluation of ROS generation and membrane potential were performed separately to exclude overlapping of the emissions after staining with H2DCFDA and DiOC6. The optimal concentration of the dyes and duration of the staining for each microalgae species were chosen based on previous works [31 (link)].
To determine the size of microalgae cells, a size calibration kit F13838 (Molecular probes, USA) with the certified size distribution of 1, 2, 4, 6, 10, and 15 μm was used for the FSC emission channel.
The number of alive microalgae cells in each measurement was determined using FSC/SSC dot cytogram (forward scattering to side scattering ratio) which allowed for the separation of the population of events with the sizes similar to the expected size of microalgae cells. Then, nonalgal events were excluded from the separated population by the absence of chlorophyll a fluorescence in the emission filter FL3 (690 nm), where all the algal cells had high MFI. Dead cells were excluded from the counting by them staining with propidium iodide (PI) according to the standard bioassay protocol [33 (link)], where dead cells obtained high MFI in the emission filter FL1 (610 nm).
The level of reactive oxygen species (ROS) generation in microalgae cells was assessed using non-fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), which activates in the presence of ROS [34 (link)]. The membrane potential of microalgae cells was assessed by a lipophilic, positively charged fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6), which is capable of binding to membranes (mitochondria and endoplasmic reticulum) and other hydrophobic negatively charged cell structures [35 (link)]. Both ROS generation and membrane potential were registered as MFI in the emission filter FL2 (525 nm) compared to the MFI of the control group in the same emission channel. The evaluation of ROS generation and membrane potential were performed separately to exclude overlapping of the emissions after staining with H2DCFDA and DiOC6. The optimal concentration of the dyes and duration of the staining for each microalgae species were chosen based on previous works [31 (link)].
To determine the size of microalgae cells, a size calibration kit F13838 (Molecular probes, USA) with the certified size distribution of 1, 2, 4, 6, 10, and 15 μm was used for the FSC emission channel.
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2',7'-dichlorodihydrofluorescein diacetate
2-(2-(2-chloro-3-(2-(3,3-dimethyl-5-sulfo-1-(4-sulfo-butyl)-3H-indol-2-yl)-vinyl)-cyclohex-2-enylidene)-ethylidene)-3,3-dimethyl-1-(4-sulfo-butyl)-2,3-dihydro-1H-indole-5-carboxylic acid
3,3'-dihexaoxycarbocyanine iodide
Biological Assay
Cells
Cellular Structures
Chlorophyll A
Dyes
Endoplasmic Reticulum
Flow Cytometry
Fluorescence
Fluorescent Dyes
Iodides
Light
Membrane Potentials
Microalgae
Mitochondria
Molecular Probes
Propidium Iodide
Reactive Oxygen Species
Tissue, Membrane
As a measure of cell viability, the cytoplasmic membrane’s integrity and mitochondrial membrane permeability changes were examined using propidium iodide (Sigma, P4170) and DiOC6 (Molecular Probes D273), respectively. Cells were pelleted and stained with 1 mg/mL PI and 50 nM DiOC6 and incubated for 30 min at room temperature to assess the incorporation of the dyes after CGA treatments. Flow cytometry was used to analyze 10,000 events with BD LSRFortessa. FlowJo was used to calculate the mean fluorescence intensity (MFI).
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3,3'-dihexaoxycarbocyanine iodide
Cell Membrane Permeability
Cells
Cell Survival
Dyes
Flow Cytometry
Fluorescence
Mitochondria
Mitochondrial Membranes
Molecular Probes
Permeability
Plasma Membrane
Propidium Iodide
Flow cytometry (FC) is a procedure that allows multiparameter analysis of the suspended cell component in an individual, cell-by-cell manner, through its physicochemical characteristics, identifying the expression of cellular proteins and any cellular component or function that can be labeled with a fluorochrome. Flow cytometry allows you to measure the different parameters of a cell. These are divided into nuclear parameters, cytoplasmic parameters, surface parameters and extracellular parameters. Phytoplanktonic microalgae, both marine and freshwater, are mostly unicellular and rarely measure more than a few tens of micrometers in diameter, making them perfect candidates for easy analysis of their integrity of cytoplasmic and mitochondrial membranes using commercial flow cytometry equipment [21 (link),22 (link),23 (link),24 ,25 (link),26 ,27 (link)]. Part of the biological material of the microalgae Scenedesmus sp. thawed by each treatment (DMSO 5%, DMSO 10%, MET 5%, MET 10%, Control) was used in parallel to perform the respective flow cytometry studies and thus determine the integrity of the cytoplasmic and mitochondrial membrane. These biological samples were processed in the laboratories of the Cellular Immunology and Immunogenetics (GICG) group of the Cytometry Unit of the Medical Research Institute of the University of Antioquia, Medellín.
The straws cryopreserved with each treatment were thawed in a serological water bath (Memmert, WNB 7–45, Baviera, Schwabach, Germany) at a constant temperature of 35 °C for 90 s. Once thawed, they were deposited in 5 mL plastic tubes. Subsequently, 100 μL was taken with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany) of the microalgae Scenedesmus sp. and deposited in another pipe, to which was added 400 μL of an enveloping solution (isotonic solution) plus 50 μL of a solution containing fluorochromes; 3,3′-dihexyloxocarbocyanine (DiOC6 (3)) (70 nm) + propidium iodide (PI) (Sigma, St. Louis, MI, USA) at a rate of 2 μg/mL with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany. This was the case for each treatment. Subsequently, the corresponding tests were performed with a FACS CANTO II CYTOMETER (BD LSRFortessa™ San José, California, USA) by excitation of a 488 nm laser and fluorescence detection at 530/30 nm and 670 nm for DiOC6 (3) and PI, respectively. In the same way, fresh samples not cryopreserved were taken as controls, and the same management was given.
The straws cryopreserved with each treatment were thawed in a serological water bath (Memmert, WNB 7–45, Baviera, Schwabach, Germany) at a constant temperature of 35 °C for 90 s. Once thawed, they were deposited in 5 mL plastic tubes. Subsequently, 100 μL was taken with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany) of the microalgae Scenedesmus sp. and deposited in another pipe, to which was added 400 μL of an enveloping solution (isotonic solution) plus 50 μL of a solution containing fluorochromes; 3,3′-dihexyloxocarbocyanine (DiOC6 (3)) (70 nm) + propidium iodide (PI) (Sigma, St. Louis, MI, USA) at a rate of 2 μg/mL with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany. This was the case for each treatment. Subsequently, the corresponding tests were performed with a FACS CANTO II CYTOMETER (BD LSRFortessa™ San José, California, USA) by excitation of a 488 nm laser and fluorescence detection at 530/30 nm and 670 nm for DiOC6 (3) and PI, respectively. In the same way, fresh samples not cryopreserved were taken as controls, and the same management was given.
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3,3'-dihexaoxycarbocyanine iodide
Bath
Biopharmaceuticals
Cellular Structures
Cytoplasm
Flow Cytometry
Fluorescence
Fluorescent Dyes
Isotonic Solutions
Marines
Microalgae
Mitochondrial Membranes
Phytoplankton
Propidium Iodide
Proteins
Scenedesmus
Sulfoxide, Dimethyl
Toxic Epidermal Necrolysis
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DiOC6 is a fluorescent dye used for the detection and analysis of mitochondrial membrane potential in cells. It is a lipophilic cationic dye that accumulates in active mitochondria due to the negative membrane potential. DiOC6 can be used in flow cytometry, fluorescence microscopy, and other fluorescence-based techniques to assess mitochondrial function and health.
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DiOC6(3) is a fluorescent dye used for staining and visualizing mitochondria in live cells. It is a lipophilic cation that accumulates in the mitochondrial membrane potential of cells.
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DiOC6 is a fluorescent dye used in cell biology research. It selectively stains the mitochondrial membrane potential of living cells.
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DMSO is a versatile organic solvent commonly used in laboratory settings. It has a high boiling point, low viscosity, and the ability to dissolve a wide range of polar and non-polar compounds. DMSO's core function is as a solvent, allowing for the effective dissolution and handling of various chemical substances during research and experimentation.
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The FACSCalibur flow cytometer is a compact and versatile instrument designed for multiparameter analysis of cells and particles. It employs laser-based technology to rapidly measure and analyze the physical and fluorescent characteristics of cells or other particles as they flow in a fluid stream. The FACSCalibur can detect and quantify a wide range of cellular properties, making it a valuable tool for various applications in biology, immunology, and clinical research.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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DiOC6(3) is a fluorescent dye used for the detection and analysis of mitochondrial membrane potential in cells. It is a cationic lipophilic dye that accumulates in the mitochondria of living cells in proportion to the membrane potential.
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3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)], is a lipophilic fluorescent dye. It can be used to stain mitochondria in live cells.