α-Naphthaleneacetic acid (NAA) was purchased from Duchefa; L-kynurenine (kyn) from Sigma (St. Louis, MO, USA); and BCECF-AM, FM4-64, MDY-64 and propidium iodide (PI) from Life Technologies (Carlsbad, CA, USA). All chemicals except PI were dissolved in dimethyl sulfoxide (DMSO). NAA and Kyn were applied in solid one-half MS medium, the dyes BCECF-AM, FM4-64 and MDY-64 in liquid one-half MS medium, and PI in distilled water.
Mdy 64
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The MDY-64 is a laboratory instrument designed for conducting various analytical tests and procedures. It serves as a versatile tool for researchers and scientists working in a wide range of fields. The core function of the MDY-64 is to perform precise measurements and analyses, enabling users to gather accurate data and insights for their research and development activities.
Automatically generated - may contain errors
Lab products found in correlation
L kynurenine kyn, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Bcecf am, by Thermo Fisher Scientific (1 mentions)
α naphthaleneacetic acid, by Duchefa Biochemie (1 mentions)
Estradiol, by Merck Group (1 mentions)
Fm4 64, by Merck Group (1 mentions)
β estradiol, by Merck Group (1 mentions)
2 7 bis 2 carboxyethyl 5 6 carboxyfluorescein acetoxymethyl ester bcecf am, by Merck Group (1 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Rhodamine 123, by Thermo Fisher Scientific (1 mentions)
Axiovert 135, by Zeiss (1 mentions)
Axioplan 2 imaging microscope, by Zeiss (1 mentions)
Carboxy dcfda, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Leica (1 mentions)
Dmi 6000 b inverted microscope, by Hamamatsu Photonics (1 mentions)
C9100 13 camera, by Hamamatsu Photonics (1 mentions)
Axioskop epifluorescence microscope, by Zeiss (1 mentions)
Calcofluor white stain, by Merck Group (1 mentions)
14 protocols using mdy 64
1
Fluorescent Dye Application Protocols
2
Fluorescent Dye Labeling Protocol
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All chemicals were dissolved in DMSO and were applied in solid ½ MS-medium. Only dyes were applied in liquid ½ MS-medium before imaging. 1-naphthaleneacetic acid (NAA) was obtained from Duchefa (Netherlands), 5-F-IAA, estradiol, FM4-64, L-kynurenine (Kyn) and propidium iodide (PI) from Sigma (MO, USA), wortmannin (WM) from Cayman Chemical (MI, USA), MDY-64 from life technologies (CA, USA) and auxinole was kindly provided by Ken-ichiro Hayashi (Hayashi et al., 2012 (link)).
3
MDY-64 Fluorescent Cell Labeling
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MDY-64 was obtained from Life Technologies. Briefly, at indicated time points, cells were resuspended at 2–4 OD600 units/mL in labeling buffer (10 mM HEPES, pH 7.4, 5% glucose). MDY-64 was then added to a final concentration of 10 µM and cells were incubated for 5 min at room temperature. Cells were then resuspended in fresh labeling buffer and analyzed by microscopy.
4
Cell Viability Assay with Chemical Compounds
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All chemicals were dissolved in dimethyl sulfoxide (DMSO) and applied in solid or liquid ½ MS medium. MDY‐64 was obtained from Life Technologies (CA, USA), β‐estradiol, propidium iodide (PI), 2′,7′‐Bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein acetoxymethyl ester (BCECF‐AM) from Sigma (MO, USA), and fusicoccin (FC) and epigallocatechin gallate (EGCG) from Cayman Chemical (MI, USA). The RALF1 peptide (mature RALF1, amino acid sequence: ATTKYISYQSLKRNSVPCSRRGASYYNCQNGAQANPYSRGCSKIARCRS) was obtained from Peptron (KOR).
5
Vacuolar Morphology and Crz1 Localization
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To evaluate vacuolar morphology, overnight cultures of JEC21, PK7, and PK50 were diluted 1:25 into fresh YPD and mock treated or treated with 2 µg/ml toremifene. After growth for an additional 4 h, 1 ml of each culture was washed with DPBS (Dulbecco's phosphate-buffered saline) and resuspended in 10 µM MDY-64 (Invitrogen), 10 mM HEPES (pH 7.4), 5% glucose. Cells were incubated in the dark at room temperature for 1 h and then placed on ice until imaging. To determine the effect of small molecules on Crz1 nuclear localization, overnight cultures of strain XW252 were diluted 1:50 in fresh YPD and treated with 1/4 the MIC of the test compound or carrier. The resulting cultures were incubated for an additional 4 h at either 30°C or 37°C. Samples were washed and resuspended in DPBS for imaging. Images were collected with a Nikon ES80 epifluorescence microscope equipped with a CoolSnap charge-coupled device (CCD) camera using NIS-Elements software and processed in PhotoShop. For quantitative analysis, at least 100 cells with clearly visible signals for both fluorophores were evaluated by eye for colocalization of Crz1 and Nop1. All imaging results were performed on multiple days from independent cultures.
6
Exogenous Sterol Utilization in Yeast
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Honokiol (5,5’-diallyl-2,4’-dihydroxybiphenyl) was obtained from Xi'an Yuquan Biological Technology Co., Ltd. and its purity was over 98% as analyzed by high-performance liquid chromatography. Yeast vacuole membrane marker MDY-64 was purchased from Invitrogen (Thermo Fisher Scientific, USA). EGTA, ergosterol, and amiodarone (AMD) were purchased from Aladdin Bio-Chem Technology Co., Ltd. (China). For analysis of exogenous sterol utilization, ergosterol was dissolved in a mixture of ethanol (50%) and Tween-80 (50%) to give a 10mM stock solution, which was used to supplement the liquid medium with a final concentration of 50 μM. The same concentration of vehicle without ergosterol was used as a control.
7
Multicolor Fluorescent Cell Staining
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Vital staining of DNA was performed by adding SYBR Green I (Invitrogen, Carlsbad CA, USA) at 1/2000 dilution. To visualize mitochondria, Rhodamine 123 (Invitrogen) was added to cell suspensions at a final concentration of 0.2 µg/mL and the stained cells were incubated for approximately 10 min at room temperature. To stain vacuoles, yeast vacuole membrane marker MDY-64 (Yeast vacuole marker sampler kit, Invitrogen) was used at the concentration of 10 µM. The stained cells were observed with a fluorescence/phase contrast microscope (Axiovert 135; Zeiss, Oberkochen, Germany) connected to a cooled charge-coupled device color camera (DP73; Olympus, Tokyo, Japan).
8
Vacuole Membrane Visualization Using MDY-64
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For the vacuole membrane analysis, MDY-64 (Invitrogen; Y7536) was used as described by the manufacturer. The cells were grown to OD600 0.5–0.6 and washed twice with deionized water. Next, the cells were resuspended at 106 cells/mL in 10 mM HEPES buffer pH 7.4 containing 5% glucose. The MDY-64 marker was added to a final concentration of 10 µM and cells were incubated at room temperature for 5 minutes. After harvesting by centrifugation, the supernatants were discarded and the pellets were resuspended in fresh 10 mM HEPES buffer pH 7.4 containing 5% glucose. Fluorescence pictures were taken with Olympus BX-51 microscope equipped with a DP-72 digital camera and cellSens Dimension software.
9
Polysaccharide Capsule and Melanin Production
10
Root Hair Staining and Imaging
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Fluorescein diacetate (FDA) staining was performed as previously described (Saedler et al., 2009 (link)). Seedlings were submerged in a solution of 40 μg FDA in water for 5 min, and then mounted on a slide. Confocal microscopic images of root hairs were obtained using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany) using 63× oil immersion objective lens after excitation of the dye at 488 nm and emission was detected between 520 and 560 nm.
MDY-64 staining was performed as described (Scheuring et al., 2015 ). Seedlings were submerged in a solution of 0.25 μM MDY-64 (Invitrogen, Y7536) in 0.5X liquid MS medium for 5 min. The seedlings were then rinsed in 0.5X liquid MS medium and mounted on a slide. Confocal images of root hairs were obtained using a 63× oil immersion objective lens after excitation of the dye at 451 nm using an Ar/Kr laser, and emission was detected at 497 nm.
MDY-64 staining was performed as described (Scheuring et al., 2015 ). Seedlings were submerged in a solution of 0.25 μM MDY-64 (Invitrogen, Y7536) in 0.5X liquid MS medium for 5 min. The seedlings were then rinsed in 0.5X liquid MS medium and mounted on a slide. Confocal images of root hairs were obtained using a 63× oil immersion objective lens after excitation of the dye at 451 nm using an Ar/Kr laser, and emission was detected at 497 nm.
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