HypoMSCs were stained green with a Green Fluorescent Cell Linker kit (Sigma Aldrich, St. Louis, MO, USA). NorMSCs were stained red with a Red Fluorescent Cell Linker kit (Sigma Aldrich). Staining was done according to the manufacturer's instructions. Then, 5 × 105 cells from each type of MSCs were injected into the cirrhosis model mice through the tail vein. Six hours after treatment, the mice were anesthetized with isoflurane and the median lobes of their livers were surgically exposed. Their internal liver surfaces were observed under an imaging system consisting of a two-photon inverted microscope (A1R-MP, Nikon, Tokyo, Japan) driven by a laser (Chameleon Vision Ti: Sapphire, Coherent) tuned to 880 nm and fitted with a water multi-immersion objective lens (Plan Fluor; N.A., 0.75; Nikon, Tokyo, Japan). Raw imaging data were processed with Imaris (Bitplane, Zurich, Switzerland).
Green fluorescent cell linker kit
Manufactured by Merck Group
Sourced in United States
The Green Fluorescent Cell Linker Kit is a laboratory product designed to label cells with a green fluorescent dye. The kit provides a method for covalently linking a fluorescent probe to the cell membrane, enabling the visualization and tracking of labeled cells.
Automatically generated - may contain errors
Lab products found in correlation
Red fluorescent cell linker kit, by Merck Group (2 mentions)
Plan fluor, by Nikon (1 mentions)
A1r mp, by Nikon (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
C57bl 6 tg ubc gfp 30scha j, by Jackson ImmunoResearch (1 mentions)
Heparin, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Phalloidin red, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Lsm 980, by Zeiss (1 mentions)
9 protocols using green fluorescent cell linker kit
1
In Vivo Tracking of Mesenchymal Stem Cells
2
Splenectomy and Splenocyte Adoptive Transfer
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Splenectomies and adoptive transfers of splenocytes in mice were performed as previously described [28 (link)]. Briefly, mice were anesthetized with isoflurane and a ~1-cm incision was made on the left side of the abdominal cavity under the rib cage. The spleen was removed by cutting the mesentery and connective tissue, and the splenic vessels were cauterized. Meloxicam (5 mg/kg, P.O.) was administered as an analgesic prior to surgery, in addition to buprenorphine (0.5 mg/kg, S.C., every 12 h for the first 48 h after surgery) and bupivacaine (0.1 ml of 0.25–0.5%, S.C., on incision site, before incision). For trafficking studies, splenocytes were harvested from GFP (green fluorescent protein) transgenic mice (C57BL/6-Tg(UBC-GFP)30Scha/J, Jackson laboratory) or C57BL/6 splenocytes labeled with green fluorescence by PKH67 (Green Fluorescent Cell Linker Kit; Sigma). The spleen was excised and minced using scissors, then pipetted in ice-cold Hanks' balanced salt solution (Life Technologies) without Ca2+ and Mg2+. The mixture passed through a 70 µm strainer and then centrifuged at 3000 rpm for 10 min at 4 °C. Erythrocytes were removed with Red Blood Cell Lysis buffer (Sigma). Isolated splenocytes (1–2 × 107) were transferred into asplenic MCAO mice via the retro-orbital venous sinus and killed 24 h after for analyses.
3
Evaluating Internalization of MGUS and MM EVs
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To evaluate cells internalization rate of MGUS and MM EVs, HVEC, and H9C2 (40,000 cell/dish of 35 mm) cells were incubated 4 h with PKH67 (Green Fluorescent Cell Linker Kit-Sigma) labeled EVs (200 μg of proteins) at 37°C. As negative controls cells were incubated with PKH67 centrifuged 2 h at 100,000 × g without EVs to exclude the presence of PKH67 aggregates. Furthermore, to block endocytosis cells were pre-incubated for 30 min at 4°C or with nocodazole 20 μM (Sigma) at 37°C. After these pre-incubations, PKH67-labeled EVs (200 μg) were added and incubated 4 h, respectively, at 4°C or in the presence of the drug (18 (link)). Intracellular uptake was tested by immunofluorescence.
To investigate the EVs uptake mediators, before cell treatment, EVs were incubated with 10 μg of Abs (sheep anti-FLCs or mouse anti-CD63) for 2 h at 4°C and afterwards EVs were ultra-centrifuged to separate unbound Abs. In alternative, serum EVs were pre-treated or not with heparin (Sigma) at a final concentration of 100 ng/ml for 30 min at 4°C. Intracellular uptake was tested by immunofluorescence and/or flow-cytometry.
To investigate the EVs uptake mediators, before cell treatment, EVs were incubated with 10 μg of Abs (sheep anti-FLCs or mouse anti-CD63) for 2 h at 4°C and afterwards EVs were ultra-centrifuged to separate unbound Abs. In alternative, serum EVs were pre-treated or not with heparin (Sigma) at a final concentration of 100 ng/ml for 30 min at 4°C. Intracellular uptake was tested by immunofluorescence and/or flow-cytometry.
4
Exosome Uptake by HUVECs Imaging
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Exosome uptake by HUVECs was analyzed, and the process was performed as follows: Exosomes were labeled with the Green Fluorescent Cell Linker Kit (PKH67, green, Sigma, USA). The labeled exosomes were filtered through a 100-kDa molecular weight cut-off hollow fiber membrane. The labeled exosomes (100 μg/ml) were incubated with HUVECs for 24 h. The cells were washed twice with 1X PBS, and then immobilized with 4% formaldehyde for 30 min. The nuclei were stained with 4′, 6-di-amidino-2-phenylindole (DAPI, blue, Solarbio, China), and the cell membrane was stained with empirical formula (phalloidin, red, Sigma, USA). Finally, the uptake of exosomes by HUVECs was imaged using ImageXpress Microconfocal with MetaXpress software (overall magnification 100 ×).
5
Cellular Uptake of Labeled sEVs
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Isolated sEVs were tested for their ability to enter cells. The sEVs were labeled with PKH67 using the Green Fluorescent Cell Linker Kit (Sigma–Aldrich, St. Louis, MO) according to the manufacturer’s protocol. The labeled sEVs were isolated using ultracentrifugation again to clear out the unbound PKH67 dye. PKH67-bound small EVs were incubated with OPLL or PLL cells for 6 h at 37 °C and then washed three times with PBS. The nuclei were counterstained with DAPI (10 μg/ml) for 20 min with Triton X-100 before the cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan). The EV-free supernatant was also incubated with PKH67 and purified to serve as a non-EV control.
6
Tracking Cell Homing in Organs
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To test the homing of injected human PBMNCs to the kidney, lung, spleen, and bone marrow, QQc or non-QQc PBMNCs were labeled with the cell tracker PKH67 before injection (Green Fluorescent Cell Linker Kit, Sigma-Aldrich, IL, USA). Cells were incubated with fluorescent PKH for 5 min at 25 °C, washed 3 times, and suspended in 0.1 mL of saline before injection.
The kidneys, lungs, and spleens harvested from the anesthetized mice were fixed overnight in 4% paraformaldehyde in a darkroom at 4 °C. Fixed tissues were processed in a graded sucrose series and preserved in optimum cutting temperature (−80 °C) until analysis. Four-µm-thick cryosections were then cut, and the number of PKH-positive cells per long axis slice was counted in each organ of 3 mice in each group (4 fields per mouse) at each time point.
To count human PKH-positive cells in bone marrow, femora harvested from the mice were immersed in 4% paraformaldehyde in a darkroom at 4 °C and cryosections were prepared for analysis using the Kawamoto method24 (link).
The kidneys, lungs, and spleens harvested from the anesthetized mice were fixed overnight in 4% paraformaldehyde in a darkroom at 4 °C. Fixed tissues were processed in a graded sucrose series and preserved in optimum cutting temperature (−80 °C) until analysis. Four-µm-thick cryosections were then cut, and the number of PKH-positive cells per long axis slice was counted in each organ of 3 mice in each group (4 fields per mouse) at each time point.
To count human PKH-positive cells in bone marrow, femora harvested from the mice were immersed in 4% paraformaldehyde in a darkroom at 4 °C and cryosections were prepared for analysis using the Kawamoto method24 (link).
7
Macrophage-Leishmania Interaction Visualization
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J774A.1 macrophages and L. infantum promastigotes were stained with PKH26 dye (Red Fluorescent Cell Linker Kit, Sigma-Aldrich) and PKH67 dye (Green Fluorescent Cell Linker Kit, Sigma-Aldrich), respectively. The manufacturer' protocols were followed for staining. Briefly, a total of 107 cells mL−1 were centrifuged (400 × g ) for 5 min into a loose pellet. Pellet was collected and resuspended with 1 mL of diluent to prepare a 2× cell suspension. Prior the staining, 2× dye solution (4 × 10−6 M) in Diluent C by adding 4 μL of the PKH ethanolic dye solution to 1 mL of Diluent C were prepared in a falcon tube. Afterwards, 1 mL of 2× dye solution were mixed with 1 mL of 2× cell suspension to obtain 1 × 107 cells mL−1 and 2 × 10−6 M PKH26 dye. After incubation of the cell/dye suspension for 1–5 min, the staining was stopped by adding an equal volume (2 mL) of serum and incubated for 1 min to allow binding of excess dye. Cells were washed following two more centrifugation steps (400 × g for 10 min) to ensure removal of unbound dye and then resuspended in a complete medium. Cells were fixed in 2% paraformaldehyde by treatment for 15 min. Prior the infection, the promastigotes and macrophages were examined by flow cytometry (CytoFLEX S, Beckman Coulter, USA) with a total of 100 000 events to ensure that all cells are successfully stained with PKH dyes and can be detected.
8
Tracing Exosome Incorporation in Canine COCs
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To trace the incorporation of OC-Exo into COCs during IVM, exosomes were labeled with a green fluorescent reagent (PKH67, Green Fluorescent Cell Linker Kit, Sigma Chemical Co.) as per the manual for 24, 48, and 72 h of IVM. In brief, OC-Exo pellets were resuspended in diluent C and PKH67 solution. After incubating for 5 min at RT, the labeling action was stopped by incubating for 1 min with 1 mL of exosome-free FBS. Then, the IVM medium was added and centrifugation was performed at 100,000 g for 30 min at 4°C. After discarding the supernatant, the pellets were resuspended in IVM medium supplemented with 10% exosome-free FBS and stored at -30°C. The COCs were cultured for 72 h, while labeled exosomes were incubated with COCs for 24, 48, and 72 h, respectively. Exosome incorporation by COCs was evaluated under a fluorescence microscope. In total, 48 canine COCs were randomly selected and used in four independent replicates for the incorporation of labeled OC-Exo experiment and four COCs were used per repeat (control group: 16, 25 µg/mL OC-Exo group: 16, 50 µg/mL OC-Exo group: 16).
9
Immunofluorescence Staining of Mycobacterium leprae
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SCs in 12-well plates were fixed using 4% paraformaldehyde for 1 hour followed by permeabilization with 0.5% Triton X-100 for 10 min. After blocking with 5% BSA for 1 h, cells were incubated with the primary antibodies at 37°C for 2 h and secondary antibodies at room temperature for 1 h. Cell nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole) for 5 min, and stained cells were imaged using a confocal laser scanning microscope (LSM980, Zeiss, Oberkochen, Germany). M. leprae were stained with PKH67 dye according to the manufacturer’s instructions (Green Fluorescent Cell Linker Kit, Sigma-Aldrich, St Louis, MO, USA) before infection.
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