The assay of vacuolar accumulation of the cell-tracker blue dye (Invitrogen), CMAC, was optimized for use in a 96-well format. In brief, cells were grown and diluted as described for the pigmentation assay with SC5314 and YNB as the tested strain and medium. For the dose-response assay, serial dilutions of the compound stocks were made in order to keep the final concentration of DMSO at 0.5%. Cells were incubated at 30°C in the presence of compounds or DMSO for 24 h. CMAC was added at a final concentration of 25 μM. After mixing, plates were incubated at room temperature in the dark for 30 min. CMAC accumulation was then measured from cell pellets. Fluorescence was measured at 354 nm excitation and 469 nm emission with a 20 nm bandwidth and growth quantified as OD600nm using a Biotek Synergy imaging plate reader.
Celltracker blue dye
Manufactured by Thermo Fisher Scientific
CellTracker Blue dye is a fluorescent cell-permeant dye used for tracking and visualizing cells in various applications. It provides a blue-fluorescent signal upon entering live cells and can be used to label and monitor cell populations.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
35 mm collagen coated glass bottom culture dish, by MatTek (2 mentions)
Opti mem 1 medium, by Thermo Fisher Scientific (2 mentions)
Synergy imaging plate reader, by Agilent Technologies (1 mentions)
Fluorescence microscopy, by Nikon (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
H2dcfda, by Nikon (1 mentions)
Celltracker orange dye, by Thermo Fisher Scientific (1 mentions)
F 2500, by Hitachi (1 mentions)
Ritc dextran, by Merck Group (1 mentions)
Pm100d optical power meter, by Thorlabs (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Lab tek 2 chamber, by Thermo Fisher Scientific (1 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Eclipse ti2 inverted microscope, by Nikon (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
10 protocols using celltracker blue dye
1
Vacuolar Accumulation Assay for CMAC
2
Mitochondrial Activity and Oxidative Stress in Oocytes
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To assess ΔΨm, denuded MII-stage oocytes were incubated with 2 μM JC-1 (Invitrogen, Waltham, MA, USA) for 1 h at 37.5°C in the dark. The ΔΨm of oocytes was then calculated as the ratio of
red fluorescence intensity (J-aggregates; corresponding to activated mitochondria) to green fluorescence intensity (J-monomers; corresponding to inactive mitochondria) using ImageJ software.
The fluorescence intensity of the resulting oocytes was analyzed using a fluorescence microscope (Nikon). ROS levels were measured by a 2′,7′-dichlorofluorescein assay (H2DCFDA; Thermo
Fisher Scientific, Waltham, MA, USA). In brief, denuded MII-stage oocytes were cultured in 0.1% BSA-PBS containing 10 μM H2DCFDA for 15 min at 37.5°C in the dark, and then visualized at an
excitation of 485 nm and emission of 535 nm. GSH levels were quantified with the CellTracker™ Blue dye (4-chloromethyl-6, 8-difluoro-7-hydroxycoumarin, CMF2HC; Invitrogen). In brief, denuded
MII-stage oocytes were incubated in 0.1% BSA-PBS medium containing 10 μM CMF2HC for 15 min at 37.5°C in the dark, and then visualized at an excitation of 371 nm and emission of 464 nm. The
fluorescence intensity (1 sec after the shutter opening with 10 msec exposure for H2DCFDA; 3 sec after the shutter opening with 100 msec exposure for CMF2HC) of the resulting oocytes was
analyzed by fluorescence microscopy (Nikon) using ImageJ.
red fluorescence intensity (J-aggregates; corresponding to activated mitochondria) to green fluorescence intensity (J-monomers; corresponding to inactive mitochondria) using ImageJ software.
The fluorescence intensity of the resulting oocytes was analyzed using a fluorescence microscope (Nikon). ROS levels were measured by a 2′,7′-dichlorofluorescein assay (H2DCFDA; Thermo
Fisher Scientific, Waltham, MA, USA). In brief, denuded MII-stage oocytes were cultured in 0.1% BSA-PBS containing 10 μM H2DCFDA for 15 min at 37.5°C in the dark, and then visualized at an
excitation of 485 nm and emission of 535 nm. GSH levels were quantified with the CellTracker™ Blue dye (4-chloromethyl-6, 8-difluoro-7-hydroxycoumarin, CMF2HC; Invitrogen). In brief, denuded
MII-stage oocytes were incubated in 0.1% BSA-PBS medium containing 10 μM CMF2HC for 15 min at 37.5°C in the dark, and then visualized at an excitation of 371 nm and emission of 464 nm. The
fluorescence intensity (1 sec after the shutter opening with 10 msec exposure for H2DCFDA; 3 sec after the shutter opening with 100 msec exposure for CMF2HC) of the resulting oocytes was
analyzed by fluorescence microscopy (Nikon) using ImageJ.
3
Imaging Amoeboid-CHO Cell Interactions
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Approximately 5 × 105 transformants were cultured on a 35 mm collagen-coated glass-bottom culture dish (MatTek Corporation, Ashland, MA) in 3 ml of BI-S-33 medium under anaerobic conditions. CHO cells were stained for 30 min with 20 mM CellTracker blue dye or CellTracker orange dye (Molecular probes, Eugene, OR) in F12 medium containing 10% FCS. After staining, CHO cells were washed three times with fresh F12 medium, and ~2 × 105 CHO cells in 200 μl F12 medium were added to the GFP-tagged protein-expressing amoeba in a glass-bottom dish. The culture was carefully covered with a coverslip, and overloaded medium was removed. The junction of the coverslip and slide glass was sealed with nail polish, and the culture was incubated at 35 °C in a temperature control unit on Zeiss, LSM780 equipped with a ×63/1.4 oil immersion objective and CCD camera.
4
Quantifying CHO Cell Destruction by E. histolytica
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The destruction of CHO monolayers was quantified as described previously [63 (link)] with slight modifications. Briefly, CHO cells were labelled with 40 μM of Cell tracker blue dye (Molecular probes, Eugene, OR) in F12 medium containing 10% FCS. After staining, labelled CHO cells were washed three times with fresh pre-warmed F12 medium. Approximately, 1.5×105E. histolytica transformants were mixed with pre-warmed OPTI-MEM I medium (Life Technologies) supplemented with 5 mg/ml L-cysteine and 1 mg/ml ascorbic acid (transfection medium) pH 6.8, and added over labelled CHO cells and incubated at 35°C for 0, 60 and 120 min. After incubation, the remaining CHO cells were collected using trypsin and the fluorescence of Cell tracker blue was measured using a fluorometer (F-2500, Hitachi, Japan) with excitation and emission at 353 and 465 nm respectively. The number of adherent CHO cells was proportional to the intensity of Cell tracker blue staining and expressed as a percentage of the remaining fluorescence of untreated CHO cells.
5
Visualizing Pinocytosis in Entamoeba histolytica
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Approximately 5×105 transformants were cultured on a 35mm collagen-coated glass- bottom culture dish (MatTek Corporation, Ashland, MA) in 3 ml of BI-S-33 medium under anaerobic conditions. CHO cells were stained for 30 min with 40 μM Cell tracker blue dye (Molecular probes, Eugene, OR) in F12 medium containing 10% FCS. After staining, CHO cells were washed three times with fresh F12 medium, and approximately 2 × 105 CHO cells in 200 μl F12 medium were added to the GFP tagged protein expressing amoeba in a glass-bottom dish. Transfection medium (TM) [OPTI-MEM I medium (Life Technologies) supplemented with 5 mg/ml L-cysteine and 1 mg/ml ascorbic acid] containing the fluorescent fluid-phase marker RITC-dextran (2 mg ml-1; MW = 70 000; Sigma-Aldrich, Japan) were added to the GFP tagged protein expressing amoeba to study the pinocytosis process in live E. histolytica trophozoites. The culture was carefully covered with a coverslip, and overloaded medium was removed. The junction of the coverslip and slide glass was sealed with nail polish, and the culture was incubated at 35°C in a temperature control unit on Zeiss, LSM780 equipped with a 63x/1.4 oil immersion objective and CCD camera.
6
AvIR-Mediated Phototoxicity Assessment
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The phototoxic effect of AvIR was assessed by using the LIVE/DEAD Cell Imaging Kit (Thermo Fisher Scientific). Cells were seeded onto an 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific) at a density of 10,000 cells/well 1 day before AvIR-PIT. The next day, the cells were treated by adding biotinylated anti-CEA (Bio-CEA), biotinylated anti-EpCAM (Bio-EpCAM) (5 µg/ml), or an equal volume of DPBS for 30 min, followed by adding AvIR (5 µg/ml) with another incubation for 30 min. The cells were exposed to NIR light (3 J/cm2) from a light-emitting diode (LED) light source (Shiokaze Giken, Niigata, Japan), which emits red light with a peak at 690 nm. The irradiation energy density was measured with a PM100D optical power meter (Thorlabs, Tokyo, Japan). The irradiated cells were incubated with a mixture of Live Green and Dead Red solutions for 20 min and were subsequently imaged using a fluorescence microscope BZ-9000 (Keyence, Osaka, Japan). To assess the target specificity, AvIR-mediated PIT was also performed for co-cultured CHO-CEA and CHO-EpCAM cells. The CHO-CEA cells were stained with CellTracker Blue dye (Thermo Fisher Scientific) and were co-cultured with unlabeled CHO-EpCAM cells in a Lab-Tek II chamber on the day before AvIR-PIT.
7
Confocal Microscopy of Platelet Aggregate
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A confocal microscope (C2+, Nikon, Tokyo, Japan) was employed on the analysis of platelet aggregate morphology. Same conditions of treatment dosage for ND and cell death inhibitors were applied in the confocal microscopy as the conditions used in the platelet cell death analyses. To distinguish populations of platelets, NDs and platelet-ND aggregates, CellTracker Blue Dye (ThermoFisher Scientific) labeled mouse platelets, and red fluorescent 50 nm NDs (brFND-50, FND Biotech) were used in this experiment. The counts of platelet aggregates per field (> 400 pixels) and the total platelet aggregate area (pixels) per field were analyzed using ImageJ software (version 1.32; National Institutes of Health, USA) (38 (link), 68 (link)).
8
Live-Dead Cell Cytotoxicity Imaging Assay
9
Endolysosomal Imaging Using Confocal Microscopy
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Cells were grown to mid-log phase and resuspended in fresh synthetic complete (SC) media prior to imaging experiments. Cells were loaded to a glass slide and imaging was performed using a Zeiss 980 laser-scanning confocal instrument equipped with an Airyscan2 detector and a 63× zoom Plan-Apochromat objective lens with 1.4 Numerical Aperture (Carl Zeiss AG, Oberkochen, Germany). YPD containing 1 μM FM4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) dye (Thermo Fisher Scientific) followed by washes in SC media was used to label the endolysosomal system, with chases performed in media lacking dye for either 5 or 30 min. SC media containing 50 µM CMAC (7-amino-4-chloromethylcoumarin) CellTracker Blue dye (Thermo Fisher Scientific) was used to label the vacuolar lumen. Blue, green, and red fluorescence was excited using solid-state lasers, using excitation wavelengths of 405 nm, 488 nm, and 561 nm, respectively, with emissions measured at wavelengths of 422/497 nm, 460/550 nm, and 570/620 nm, respectively.
10
Oocyte ROS and GSH Quantification
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To measure ROS levels, oocytes were incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Thermo Fisher Scientific, Waltham, USA) for 30 min, followed by spectroscopic
analysis (green fluorescence, UV filters, 490 nm). To measure GSH levels, oocytes were incubated with 10 μM Cell Tracker Blue dye 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC)
(Thermo Fisher Scientific) for 30 min, followed by spectroscopic analysis (blue fluorescence, UV filters, 370 nm). The fluorescence intensity of the oocytes was analyzed using ImageJ
software.
analysis (green fluorescence, UV filters, 490 nm). To measure GSH levels, oocytes were incubated with 10 μM Cell Tracker Blue dye 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC)
(Thermo Fisher Scientific) for 30 min, followed by spectroscopic analysis (blue fluorescence, UV filters, 370 nm). The fluorescence intensity of the oocytes was analyzed using ImageJ
software.
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