EVs isolated from non-labeled hBM-MSCs were tagged with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St Louis, MO, USA) at room temperature (RT) for 5 min in the dark and blocked with fetal bovine serum (FBS), according to manufacturer’s instructions. The unincorporated labels were removed by EV centrifugation at 100,000×g for 75 min at 4 °C using a Thermo Scientific T-865 Fixed Angle Rotor Thermo Scientific Sorvall WX Ultracentrifuge Series. EVs were washed with DPBS and subjected to additional centrifugations. Then, the pellet was re-suspended in 1 ml DPBS for further use.
Red fluorescent cell linker kits mini26
Manufactured by Merck Group
Sourced in United States
The Red Fluorescent Cell Linker Kits MINI26 are a set of labeling reagents designed for fluorescent labeling of live cells. The kits contain a red fluorescent dye that can be easily incorporated into the cell membrane, enabling visualization of the labeled cells.
Automatically generated - may contain errors
Lab products found in correlation
Sorvall wx ultracentrifuge series, by Thermo Fisher Scientific (1 mentions)
T 865 fixed angle rotor, by Thermo Fisher Scientific (1 mentions)
100 mm2 tissue culture dish, by Jet Biofil (1 mentions)
Ca and mg, by Lonza (1 mentions)
Centrifuge 5804 r, by Eppendorf (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
6 protocols using red fluorescent cell linker kits mini26
1
Labeling of Extracellular Vesicles from hBM-MSCs
2
Evaluating BBT-EV and MSC Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the BBT-EV and MSC interaction, a 2D chemotaxis assay was developed using PKH26-labeled BBT-9 and BBT-18 according to the manufacturer’s instructions. Briefly, the labeling of cells with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St Louis, MO, USA) was performed for 5 min at RT in the dark and blocked with FBS. The unincorporated stains were removed by BBT centrifugation at 400× g for 10 min at RT using Heraeus Biofuge Primo R Centrifuge. The BBTs were washed with PBS and subjected to additional centrifugation. The pellet was resuspended, and labeled cells were plated in a 100 mm2 tissue culture dish (JetBiofil, Guangzhou, China) and incubated in an atmosphere of humidified air and 5% CO2 at 37 °C for 72 h. Culture media were supplemented with 10% of EV-depleted FBS. EVs were then isolated by SEC from BBT conditioned media. A cell migration analysis by agarose spot assay with labeled EVs from BBT was carried out following the procedures of the 2D chemotaxis assay described below in materials and methods. A control with BBT-EVs from unlabeled BBT was performed. After O/N incubation, MSCs were visualized in fluorescence microscopy.
3
Labeling hBM-MSCs with Molday ION and PKH26
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The labeling of cells with Molday ION consisted of SPIO nanoparticles, and rhodamine purchased from BioPAL (Worcester, MA, USA) was performed as previously described by us.35 (link) Briefly, 100 μL of Molday ION was added to the 5×105 hBM-MSCs cultured in 10 mL of MSCGM and incubated over 16 hours at 37°C in a humidified atmosphere containing 5% CO2. After that, medium with label was removed, cells were washed with phosphate-buffered saline (PBS), fresh medium was added, and cells were cultured for 48 or 72 hours. The labeling of cells with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St Louis, MO, USA) was performed at room temperature (RT) for 5 minutes in the dark and blocked with fetal bovine serum (FBS), according to manufacturer’s instructions. The unincorporated stains were removed by hBM-MSCs centrifugation at 400× g for 10 minutes at 20°C–25°C using Eppendorf Centrifuge 5804R. hBM-MSCs were washed with Dulbecco’s PBS (DPBS) without Ca++ and Mg++ (Lonza) and subjected to additional centrifugation. The pellet was re-suspended, and cells were plated in 75 cm2 polystyrene tissue culture flasks as described earlier.
4
Fluorescent Labeling of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EVs were labeled with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St. Louis, MO, USA), according to the manufacturer’s protocol, with slight modifications. Briefly, tick hemolymph EVs (30 μg) and rabbit serum EVs (30 μg) were resuspended in 1 ml Diluent C, respectively. A 2 × Dye Solution was prepared in Diluent C by adding 4 μl PKH26 ethanolic dye solution to 1 ml Diluent C in a centrifuge tube and mixing well. The EV suspension was mixed with the stain solution (1:1 dilution EVs/dye) and incubated for 5 min at room temperature in a dark room. The labeling reaction was stopped by adding an equal volume of 1% BSA/fetal bovine serum. Labeled EVs were ultracentrifuged at 120,000 × g for 90 min, washed with PBS and ultracentrifuged again.
5
Extracellular Vesicles Isolation from hAD-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EVs isolation was performed from the growth medium MSCGM™ of hAD-MSCs. In detail, hAD-MSCs (10 × 106 cells, passages 4–6) were cultured in 75 cm2 tissue flasks to reach 40% confluence; the culture medium was changed and the cells were incubated for additional 48–72 h to reach the confluence of 70–80%. Cell culture supernatants were collected and centrifuged at 4 °C for 10 min at 200 g and then for 10 min at 500 g. Culture supernatants were thawed and spun down vertically at 4 °C for 20 min at 2000g and then centrifuged horizontally at 100,000g for 75 min. Finally, the supernatant was discarded and the exosome pellet was re-suspended in 1 mL of PBS. The isolated EVs were marked with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St Louis, MO, USA) for 5 min at room temperature in dark room and blocked with fetal bovine serum, according to manufacturer’s instructions. Unincorporated labels were removed by exosomes centrifugation at 4 °C for 75 min at 100,000g. Finally, exosomes were washed with DPBS and subjected to additional centrifugations.
6
Exosome and Slide Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples (exosomes and slide) were previously fixed for 10 min in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Before staining the samples, they were washed 3 times on PBS and incubated for 1 h at room temperature in PBS solution plus 3% bovine serum albumin (BSA, Sigma-Aldrich). After subsequently washing the samples, they were incubated at 4 °C overnight with primary mouse antibodies anti-human CD 81, CD63, (Thermo Fisher Scientific, Waltham, MA, USA) and with fluorescent secondary goat antibodies anti-human (Alexa Fluor 555 dye, Thermo Fisher Scientific). Additional staining was also performed. Next, the exosomes were marked with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St. Louis, MO, USA) for 5 min at room temperature in a dark room and blocked with fetal bovine serum, according to the manufacturer’s instructions [49 (link)].
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!