Pre-formed biofilms (24-h-old) of PaCF1 and PaCF11 treated with LP for 24 h (as described in Section 4.7 ) were labeled with 0.4% PHK26 (Merck, Milan, Italy), an orange fluorescent lipophilic dye, according to the manufacturer’s instructions. After washings to remove unbound PHK26, biofilms were incubated in 200 μL of Calcofluor white (Merck, Milan, Italy), at a concentration of 500 μg/mL in KOH 10% for 1 min, to stain β-polysaccharides. After further washes with PBS to remove the excess of Calcofluor white, biofilms were kept protected from light until imaging. Biofilms were visualized by using the Operetta CLS High-Content Analysis System (PerkinElmer Inc., Boston, MA, USA), acquiring 29 plane confocal images at 63× magnification for each biofilm. Images were then analyzed by Harmony software (Version 4.9, Perkin Elmer Inc., Boston, MA, USA).
Phk26
Manufactured by Merck Group
Sourced in United States, Italy
PHK26 is a laboratory equipment product manufactured by Merck Group. It is a device used for performing various experiments and analyses in a controlled laboratory environment. The core function of PHK26 is to provide precise and reliable measurements or observations as required by the research or testing protocol.
Automatically generated - may contain errors
Lab products found in correlation
Diluent c, by Merck Group (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Operetta cls high content analysis system, by PerkinElmer (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Axiovert microscope, by Zeiss (1 mentions)
Phk67 green, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Vectashield h 1200, by Vector Laboratories (1 mentions)
Lsm 900, by Zeiss (1 mentions)
Magnafire, by Olympus (1 mentions)
24 well plate, by Corning (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Streptavidin apc, by Thermo Fisher Scientific (1 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Rat igg2a, by Thermo Fisher Scientific (1 mentions)
Rat igg2b, by Thermo Fisher Scientific (1 mentions)
Hamster igg, by Thermo Fisher Scientific (1 mentions)
13 protocols using phk26
1
Fluorescent Imaging of Biofilm Composition
2
Kio-CiPSLCs Chimera Formation in Zebrafish
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The cell preparation and chimera formation were conducted according to Peng et al.'s methods (Peng et al., 2019 (link)). The Kio-CiPSLCs were first collected in Diluent C (Sigma, United States) with a density of 107cells/mL. The cells were stained with PHK26 (Sigma, United States) fluorescent dye for 3–5 min 1%BSA was added to terminate the labeling reaction, and the unbound dye was removed by repeated washing with PBS. Chimera formation was evaluated by transplantation PHK26-labeled Kio-CiPSLCs into the mid-blastula stage of zebrafish embryos, as previously described (Peng et al., 2019 (link)). The labeled Kio-CiPSLCs were suspended in PBS. Approximately 200–300 donor cells (equal ratio of cells) were injected into the deep cell layer of each embryo. The recipient embryos were incubated in E3 medium at 28.5°C after injection and were monitored under a fluorescent microscope. As previously described, specific methods were carried out (Peng et al., 2019 (link)).
3
Exosome Labeling and Cellular Uptake
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The exosomes were labeled with PHK26 (Sigma, USA) and were incubated with bEnd.3 in DMEM supplemented with 10% FBS for 48 hours. Then, they were washed with PBS to stop the cell absorption and fixed in 4% paraformaldehyde. Finally, the outcome of cell absorption was observed using the Olympus BX41 microscope equipped with a CCD camera.
4
Tracking Differentiation of C2C12 Cells
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C2C12 expressing the indicated shRNAs or plasmids were stained with lipophilic cell tracking dyes PHK26 (red, pseudo-colored purple) or PHK67 (green) (Sigma-Aldrich). Staining was conducted according to the manufacturer’s protocol. Cell were mixed at a ratio 1:1 and plated in 6-well plates. Differentiation was induced the next day for 48 h. Images were acquired with an Axiovert microscope (Zeiss) at an objective of ×20. The images were analyzed using the Volocity software.
5
Chimera Formation via Labeled ziPSC Transplantation
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The ziPSCs were first trypsinized and collected at density of 107 cells/mL in Diluent C (Sigma). The cells were stained with the fluorescent dye PHK26 (red; Sigma) for 5 min. Serum was added to terminate the labeling reaction, and the unbound dye was removed by repeatedly washing with PBS.
Chimera formation was evaluated through the transplantation of PHK26-labeled ziPS cells into the midblastula stage of zebra fish embryos, as previously described 31 (link). The labeled ziPS cells were suspended in PBS. Approximately 200-300 donor cells (equal ratio of cells) were injected into the deep cell layer of each embryo. The recipient embryos were incubated in E3 medium at 28.5°C after injection and were monitored under a fluorescent microscope.
Chimera formation was evaluated through the transplantation of PHK26-labeled ziPS cells into the midblastula stage of zebra fish embryos, as previously described 31 (link). The labeled ziPS cells were suspended in PBS. Approximately 200-300 donor cells (equal ratio of cells) were injected into the deep cell layer of each embryo. The recipient embryos were incubated in E3 medium at 28.5°C after injection and were monitored under a fluorescent microscope.
6
Cell-Cell Conjugation Dynamics
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7
Angiogenesis Tube Formation Assay
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Matrigel was plated on 35 mm Ibidi μ dishes. C1 clone derived spheres labeled with the green fluorescent tracer, calcein-AM, were mixed with SVEC4-10 cells that had been stained with a red fluorescent cell tracer dye, PHK26 (Sigma-Aldrich). Nuclei were counterstained with Hoechst33342. The cellular mixture was seeded onto Matrigel plated dishes in DMEM containing 2% FBS and 5% Matrigel. Tube formation was recorded using time-lapse immunofluorescence confocal microscopy.
8
Fluorescent Labeling and Uptake of Extracellular Vesicles
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EVs were labeled using the PHK26 (Sigma-Aldrich). Briefly, 20 μg EVs were mixed with 1 mL PHK26 staining solution for 20 min, rinsed with PBS, and then centrifuged at 1,000 g for 70 min. Following the co-culture of PHK26-labeled EVs with HCAECs for 24 h, the uptake of EVs by HCAECs was observed under a confocal fluorescence microscope (Zeiss) [50 (link)].
9
Osteoblast Morphology on Biomaterial Surfaces
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We cultured human osteoblasts on each sample surface, seeded at a density of 1.5 × 104 cells. The cells were stained using PHK-26 (MINI26; Sigma-Aldrich, USA) and cultured on the sample surfaces. To stain the nuclei blue, a mounting medium with DAPI (4', 6-diamidino-2-phenylindole) (VECTASHIELD, H-1200; Vector Labs, USA) was used.
After 4 h of incubation, cell morphology was observed using a confocal laser scanning microscope (LSM900; Carl Zeiss, Germany).
After 6 days of culture, the cells were washed thrice with PBS and fixed with 3.7% (v/v) paraformaldehyde at room temperature for 15–20 min. The preserved cells were irrigated with PBS thrice, treated with 0.1% Triton X-100 for 10 min, and irrigated again with PBS. Rhodamine-phalloidin (Molecular Probes, USA) was diluted to 1:100 and left to react for 1 h at 37 °C without any light exposure. It was then sprayed with PBS three times, mounted in aqueous mount and examined using a confocal microscope.
After 4 h of incubation, cell morphology was observed using a confocal laser scanning microscope (LSM900; Carl Zeiss, Germany).
After 6 days of culture, the cells were washed thrice with PBS and fixed with 3.7% (v/v) paraformaldehyde at room temperature for 15–20 min. The preserved cells were irrigated with PBS thrice, treated with 0.1% Triton X-100 for 10 min, and irrigated again with PBS. Rhodamine-phalloidin (Molecular Probes, USA) was diluted to 1:100 and left to react for 1 h at 37 °C without any light exposure. It was then sprayed with PBS three times, mounted in aqueous mount and examined using a confocal microscope.
10
In Vivo Tracking of Labeled Extracellular Vesicles
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EVs were pre-labeled with PHK26 (Sigma-Aldrich) and incubated in the dark at 37 °C for 15 min. Then, EVs were centrifuged at 16,000×g for 60 min to remove the supernatant. Following PBS rinsing three times, the labeled EVs were injected into rats. EVs in vivo were observed under an Olympus BX41 microscope equipped with a charge-coupled device camera (Magnafire, Olympus, Tokyo, Japan) [58 (link)].
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