Paraformaldehyde 16% (Electron Microscopy Sciences, Hatfield PA), fibronectin, poly(allylamine hydrochloride), bovine serum albumin (BSA), Triton X-100, PKH 26 red dye, guinea pig tissue transglutaminase 2 (TG2), PKH 67 green dye, Texas-red maleimide (all from Sigma, St. Louis MO), Dulbecco’s phosphate buffered saline (DPBS; Mediatech, Manassas VA), Trypsin-EDTA 0.25%, Dulbecco’s modified Eagle medium (DMEM) media with 10% FBS supplement, HepG2 cells (all from ATCC, Manassas VA), human umbilical vein endothelial cells (HUVEC; Lonza, Walkersville, MD), mouse antifibronectin antibody (clone FN12−8, Takara Bio Inc., Shiga, Japan), AlexaFluor 488 goat antimouse IgG (H+L), H33342 (all from Invitrogen, Carlsbad CA), Poly(dimethylsiloxane) (PDMS; Sylgard 184, Dow Corning, Midland, MI), SU-8 photoresist and developer (MicroChem Corp, Newton, MA), and glass-bottomed dishes (MatTek, Ashland, MA).
Pkh26 red dye
Manufactured by Merck Group
Sourced in United States
PKH26 red dye is a fluorescent cell linker used for labeling and tracking living cells. It is a lipophilic, red-orange fluorescent dye that binds to the cell membrane and is inherited by daughter cells during cell division. The dye has excitation and emission wavelengths of 551 nm and 567 nm, respectively.
Automatically generated - may contain errors
Lab products found in correlation
F actin, by Yeasen (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Yeasen (2 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Sylgard 184, by Dow (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Poly allylamine hydrochloride, by Merck Group (1 mentions)
Glass bottomed dishes, by MatTek (1 mentions)
Su 8 photoresist and developer, by MicroChem (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Fitc phalloidin, by Solarbio (1 mentions)
Lsm 880, by Zeiss (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Eclipse ci l, by Nikon (1 mentions)
6 protocols using pkh26 red dye
1
Fabrication and Characterization of Cell-ECM Constructs
2
Exosome Labeling and Cellular Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tracking of the purified exosomes was accomplished by staining their membranes using PKH26 red dye (Sigma‐Aldrich). Washing of the labelled exosomes was made thrice using PBS, and exosomes were recollected by ultracentrifugation. HUVEC uptake of the labelled exosomes was evaluated by combining exosomes and cells, and the uptake was quantified using immunofluorescence.
3
Exosome Uptake Visualization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Uptake of exosomes was observed by fluorescence. First, CD34+-Exos and miR-26a-CD34+-Exos were labeled with PKH26 red dye (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer’s protocol. Then, exosomes were ultracentrifuged at 110,000×g for 70 min and the supernatant was discarded. The pellet was resuspended in 500 μL dilution C, and 4 μL PKH26 dye was dissolved in another 500 μL dilution C. The two tubes of dilution C were mixed and co-incubated for 5 min at room temperature to obtain exosomes-PKH26. The exosomes-PKH26 were then co-cultured with BMSCs and HUVECs at a concentration of 20 μg/mL. After 12 h, cells were fixed with 4% paraformaldehyde (Well, Shanghai, China) for 15 min, treated with 0.1% Triton X-100 (Beyotime) for 5 min for membrane penetration, stained with DAPI for 5 min, and washed with PBS three times. Photographic images were acquired using a fluorescence microscope (Olympus IX 70).
4
Visualizing BMSC-Derived Exosome Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A BMSCs-Exos uptake assay was conducted using PKH26 red dye (Sigma-Aldrich, USA). Briefly, BMSCs-Exos were marked with PKH26 according to the instruction book. The NP cells were incubated with PKH26-labeled exosomes. Then, cells were fixed with 4% paraformaldehyde. FITC phalloidin (Solarbio, China) was used to observed labeled the cytoskeleton of NP cells. The NP nuclei were stained with Hoechst 33342. The cells were observed with confocal microscopy (Nikon A1, Japan).
5
Visualizing Exosome Uptake in TNBC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TNBC cells were seed onto a 6 well plate overnight. After that, the PKH26 red dye (Sigma-Aldrich, St. Louis, MO, USA) was used to label exosomes. Later on, TNBC cells were treated with PKH26-labeled exosomes (50 μg/mL) for 24 h. Phalloidin (1:1000 dilution in 1%BSA; YEASEN Biotechnology, Shanghai, China) was used to stain actin filaments (F-actin) at 37°C for 30 min, and DAPI (10 μg/mL; YEASEN Biotechnology) was used to stain cell nuclei at room temperature for 5 min. Next, exosomes absorbed by TNBC cells were captured under a confocal laser scanning microscope (ZEISS LSM880, light: laser; objective: 40x; Zeiss, Jena, Germany). Green fluorescence, excitation wavelength/emission wavelength: 490 nm/530 nm; blue fluorescence: 364 nm/454 nm; red fluorescence, 540 nm/615 nm.
6
Exosome Internalization and Apoptosis in TNBC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TNBC cells were seed onto a 6 well plate overnight. After that, the PKH26 red dye (Sigma-Aldrich, St. Louis, MO, USA) was used to label exosomes. Later on, TNBC cells were treated with PKH26-labeled exosomes (50 μg/mL) for 24 h. Phalloidin (1:1000 dilution in 1%BSA; YEASEN Biotechnology, Shanghai, China) was used to stain actin filaments (F-actin) at 37°C for 30 min, and DAPI (10 μg/mL; YEASEN Biotechnology) was used to stain cell nuclei at room temperature for 5 min. TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics Co., Indianapolis, IN, USA). TNBC cells were fixed with 4% paraformaldehyde for 20 min and then incubated with the TUNEL reaction mixture (solution A and solution B) for 1 h in darkness at 37°C. Finally, analysis was performed by a fluorescence microscopy (Nikon, Eclipse Ci-L, light: mercury lamp; objective: 20x; Nikon, Tokyo, Japan). Green fluorescence, 490 nm/530 nm; blue fluorescence: 364 nm/454 nm. TUNEL-positive cells (green color) were counted in 3 random fields using the Image-Pro Plus software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!