All reagents and buffers were purchased from Fujifilm Wako Pure Chemical Corporation, unless otherwise noted. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, #C2759) was purchased from Sigma, and MitoTracker™ Deep Red FM (MTDR, #M22426), LysoTracker™ Green DND-26 (LTG, #L7526) and LysoTracker™ Red DND–99 (LTR, #L7528) were purchased from Invitrogen (Thermo Fisher Scientific, USA). Cell Light-Red Fluorescent Protein (RFP) reagents (Thermo Fisher Scientific) were used for staining the early endosome (#C10587), late endosome (#C10589), and lysosome (#C10589). Mem Dye-Deep Red (Dojindo laboratories, #EX03) and PlasMem Bright Red (Dojindo laboratories, #P505) were used for labeling the Extracellular Vesicles (EVs) and for staining the cellular membrane, respectively. All fluorescent dyes were used following the product manuals. Penicillin–streptomycin (#15140163, 10,000 units/mL), fetal bovine serum (FBS, #26140079), and Dulbecco’s modified Eagle’s medium (DMEM, #11965092) were all purchased from Gibco (Thermo Fisher Scientific, USA). Phosphate-buffered saline (PBS, #SH30256.01) was purchased from Hyclone (GE Healthcare Life Sciences).
Plasmem bright red
Manufactured by Dojindo Laboratories
Sourced in Japan
PlasMem Bright Red is a fluorescent dye used for labeling and visualizing the cell membrane in living cells. It has maximum excitation and emission wavelengths of 644 nm and 665 nm, respectively.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Permafluor mounting medium, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (2 mentions)
Exosparkler exosome membrane labeling kit green, by Dojindo Laboratories (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Carbonyl cyanide m chlorophenylhydrazone cccp, by Merck Group (1 mentions)
Mitotracker deep red fm, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fm4 64, by Merck Group (1 mentions)
Bz x810, by Keyence (1 mentions)
Bz h4xf, by Keyence (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
Nucspot live 650, by Biotium (1 mentions)
6 protocols using plasmem bright red
1
Multimodal Imaging of Cellular Organelles
2
Visualizing SPC-induced Cell Contraction
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We observed the morphology of SPC-induced contracting cells using the inverted microscope CKX53. Live-cell imaging was performed by first mounting HCASMCs onto glass coverslips, and staining the nuclei with DAPI (Sigma Aldrich, St. Louis, MO, USA) for >30 min after sample preparation, as mentioned in Section 2.1 . Thereafter, we stained the plasma membranes with PlasMem Bright Red (Dojindo, Kumamoto, Japan) for 5 min or endosomes with 4 μM of FM4-64 (Sigma Aldrich, St. Louis, MO, USA), which can stain in live cells. The cells were then washed with HEPES buffer, and SPC-induced contraction was induced by 30 μM of nitrobenzoxadiazole (NBD)-SPC for 10 min; that is, SPC with guaranteed fluorescence, with NBD bound to the C-6 position of SPC (Cayman, Ann Arbor, MI, USA). Since SPC is a non-fluorescent compound, we evaluated its intracellular behavior using NBD-SPC, which is commonly used to study the metabolism and transport of sphingolipids [17 (link)]. Cell staining and cross-section images of cells were obtained using an all-in-one fluorescence microscope (BZ-X810, KEYENCE, Osaka, Japan) equipped with an optical sectioning module (BZ-H4XF, KEYENCE, Osaka, Japan).
3
Fluorescent Labeling of Cells
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The micropost pattern was coated with rhodamine‐conjugated fibronectin (FNR01, Cytoskeleton Inc., Denver, CO) at a concentration of 5 µg mL−1 to promote cell adhesion while fluorescently‐labeling the microposts. For imaging F‐actin, plasma membrane, or nuclei in some experiments, cells were fixed in 4% paraformaldehyde (J61899, Alfa Aesar, Haverhill, MA) at room temperature for 15 min and washed thrice with 1X PBS (21‐040‐CM, Corning, Corning, NY). Hoechst (875756‐97‐1, Sigma‐Aldrich, St. Louis, MO) was used to stain DNA, and Alexa Fluor‐488 phalloidin (A12379, ThermoFisher Scientific, Waltham, MA) was used to stain F‐actin in fixed samples. NucSpot Live 650 (40 082, Biotium, San Francisco, CA) was used to stain DNA in live cells. PlasMem Bright Red (P505, Dojindo Molecular Technologies, Inc., Rockville, MD) was used to stain the plasma membrane. All reagents were used at the concentration recommended by the manufacturer.
4
Exosome Uptake by Macrophages
5
Visualizing MCT13 and CD147 Expression
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HEK293T cells were seeded on poly-lysine-coated culture cover glass (Matsunami Glass, Osaka, Japan) and transfected with plasmids of MCT13 and/or CD147 or GP70 by using polyethyleneimine. After 48 h of incubation, the cells were stained by PlasMem Bright Red (Dojindo laboratories, Kumamoto, Japan) and observed by fluorescence microscopy (BZ-X810). Caco-2-Tet-MCT13 cells were cultured on a Falcon cell culture insert membrane for 24 days and treated with 5 μg/mL doxycycline and 5 mM sodium butyrate for 2 days. The cells were fixed by 4% paraformaldehyde at room temperature for 10 min. The cell culture insert was mounted by using a mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and observed by fluorescence microscopy (BZ-X810).
6
Exosome Uptake by Macrophages
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GMSC-derived exosomes were labeled using an ExoSparkler Exosome Membrane Labeling Kit-Green (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocols. Then, the green fluorescence-labeled exosomes were added to PBMC-differentiated macrophages at a final concentration of 1 μg/mL and incubated for 3 h. Following incubation, cells were washed with PBS (pH 7.4), fixed with 4% PFA. Non-specific reactions were blocked with 3% BSA in PBS. Macrophages were labelled with Alexa Fluor 594 phalloidin (Thermo Fisher Scientific) for F-actin, or PlasMem Bright Red (Dojindo) for cell membrane and 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) was used for nucleus. For Coverslips were mounted using the PermaFluor Mounting medium (Thermo Fisher Scientific) and images were analyzed by LSM 700 confocal microscope (Carl Zeiss) and Zen 2012 software.
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