EVs were labeled using PKH67-GL (Sigma-Aldrich), and images were acquired using confocal microscopy. EVs were labeled for 10 min at 37 °C and the mixtures were precipitated with ExoQuick-TC reagents (System Biosciences), then incubated on ice for 30 min, centrifuged at 4 °C at 13,000 rpm, and filtered to remove unbound dyes. The pellets were suspended in 1× PBS buffer, and the labeled EVs were placed on confluent coverslips of hCMEC/D3 (Sigma-Aldrich, Millipore, CA, USA) for 24 h in a cell culture incubator at 37 °C. The labeled PKH67-Green color was imaged with a Leica SP5 Tandem Scanner Spectral 2-photon confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) with a 63× oil-immersion lens. As the negative control, PKH67-Green was prepared with all reagents, but did not include EVs, and thus ascertained for any unincorporated dyes that may be carried over after centrifugation. Nuclei were imaged by staining with Hoechst 33,342 (Sigma-Aldrich) at a concentration of 1 µg/mL in PBS for 5 min.
Pkh67gl
Manufactured by Merck Group
Sourced in United States
PKH67GL is a fluorescent labeling dye produced by Merck Group. It is designed for the long-term, stable fluorescent labeling of living cells. The dye binds to the cell membrane and is partitioned equally between daughter cells during cell division, allowing for the tracking and monitoring of cell populations.
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Lab products found in correlation
Nucblue live cell stain, by Thermo Fisher Scientific (2 mentions)
Pkh67, by Merck Group (2 mentions)
D9542, by Merck Group (2 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Hcmec d3, by Merck Group (1 mentions)
Sp5 tandem scanner spectral 2 photon confocal microscope, by Leica (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Anti cd42b apc, by Thermo Fisher Scientific (1 mentions)
C1005, by Beyotime (1 mentions)
Prism 6, by GraphPad (1 mentions)
Fv10i, by Olympus (1 mentions)
Ab104139, by Abcam (1 mentions)
Tritc phalloidin, by Yeasen (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Exo fbs 50a 1, by System Biosciences (1 mentions)
Fv3000, by Olympus (1 mentions)
Tcs sp8, by Leica (1 mentions)
μ dishes, by Ibidi (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dnase d5025, by Merck Group (1 mentions)
22 protocols using pkh67gl
1
Labeling and Imaging of Extracellular Vesicles
2
Cytotoxicity Assay with Balb/c Peripheral Cells
3
Fluorescent Uptake of Hst1 by Osteogenic Cells
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To analyze the uptake and localization of fluorescently labeled Hst1 (F-Hst1) by osteogenic cells, cells from a semi-confluent cell culture were transferred into a 48-wells plate at a density of about 3.5 × 104 cells/well and cultivated at 37°C for at least 24 h. Subsequently, the cells were washed once with DPBS (Dulbecco’s PBS, Gibco) after which serum-free medium was added. Two micrometer F-Hst1 was added and after a 1 h period, the cells were washed four times with DPBS containing 0.9 mM Ca2+ and 0.5 mM Mg2+. As a negative control, incubations without F-Hst1 were included. The cells were studied by the EVOS-FL microscope with a 20x objective with a phase contrast setting and a “Cy-5 light cube” with a 628/40 excitation filter and a 692/40 nm emission filter. Digital photographs were recorded by a computer integrated in the microscope.
To more precisely observe the uptake and localization of F-Hst1, the cells were further studied using a LEICA TCS SP8 confocal laser scanning microscopy (CLSM) system as previously described (Ma et al., 2020 (link)). Before incubation with F-Hst1, cell nuclei and membrane were stained with NucBlueTM live cell stain (Life Technologies, Grand Island, NY) and PKH67GL (Sigma–Aldrich, MO, United States) respectively, following manufacturer’s instructions.
To more precisely observe the uptake and localization of F-Hst1, the cells were further studied using a LEICA TCS SP8 confocal laser scanning microscopy (CLSM) system as previously described (Ma et al., 2020 (link)). Before incubation with F-Hst1, cell nuclei and membrane were stained with NucBlueTM live cell stain (Life Technologies, Grand Island, NY) and PKH67GL (Sigma–Aldrich, MO, United States) respectively, following manufacturer’s instructions.
4
NK-92 Cell Cytotoxicity Assay
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Cytotoxic activity of parental NK‐92 and αCD19‐CAR NK‐92 cells was evaluated by FACS analysis. Target cells (2 × 106) were pre‐stained with the green fluorescent membrane dye PKH67‐GL (Sigma‐Aldrich) and effector cells were added to 4 × 104 target cells to yield effector to target (E:T) ratios of 1:1 and 10:1. After incubation for 4 hrs, the cell mixture was centrifuged at 260 × g and stained with propidium iodide (PI, 5 μg/ml; Sigma‐Aldrich). Dead target cells were identified by simultaneous PKH67‐GL and PI ‐positive. Target cells incubated without effector cells were used to assess spontaneous cell death.
5
Erythrocyte-Platelet Interaction Imaging
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Ionomycin- or control-treated erythrocytes (109 cells) were stained using PKH67GL (Sigma Aldrich). Platelets (4x107 cells) were stained using anti-CD42b-APC (Life Technologies). Alexa Fluor®568-labelled anti-VWF (Dako) antibody was prepared using a Zenon® Alexa Fluor® 568 Rabbit IgG Labeling Kit (Molecular Probes). Erythrocytes (108 cells/ml) with or without platelets (4x106 cells/ml), anti-VWF Alexa Fluor®568 (1.2 μg/ml) and 100 μM histamine were perfused over ECs for 10 minutes. Real-time imaging was performed using the Axiovert 200M with a 40x oil objective (NA 1.3).
6
Macrophage-derived EV Uptake by RAW264.7 Cells
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PKH67 dye was added to macrophage-EV/siHMGB1 according to the kit instructions (PKH67GL, Sigma Aldrich, St. Louis, MO, USA), which was incubated at room temperature for 15 min, and then centrifuged at 1000g for 5 min, followed by removal of the supernatant. The mixture was suspended in a macrophage-EV medium and centrifuged at 1000 for 5 min. Precipitate was obtained after repeating twice, which was PKH67-labeled macrophage-EV. Then, RAW264.7 cells were incubated in the dish pre-coated with the specific cell slides. When cell confluence reached 50%, PKH67-labeled macrophage-EV/siHMGB1 was added for incubation at 37 ℃ for 24 h. The cell slides were removed, washed three times in PBS, soaked with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 2% Triton X-100 for 15 min, and then stained by 4′,6-diamidino-2-phenylindole (DAPI; 2 μg/mL, C1005, Beyotime) for 10 min. Fluorescence expression was observed by confocal microscopy.
With the similar method mentioned above, Cy3-HMGB1 was loaded to the macrophage-EV. Macrophage-EV/Cy3-siHMGB1 was then co-cultured with RAW264.7 cells for 1 h and fixed in 4% paraformaldehyde. The nuclei were stained with DAPI. Internalization of macrophage-EV by RAW264.7 cells was observed under confocal microscopy.
With the similar method mentioned above, Cy3-HMGB1 was loaded to the macrophage-EV. Macrophage-EV/Cy3-siHMGB1 was then co-cultured with RAW264.7 cells for 1 h and fixed in 4% paraformaldehyde. The nuclei were stained with DAPI. Internalization of macrophage-EV by RAW264.7 cells was observed under confocal microscopy.
7
PCSK9-Mediated Monocyte-Endothelial Adhesion Study
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THP-1 cells were individually subjected to knockdown procedures utilizing either CTRL siRNA or CAP1 siRNA, and the cells were allowed to incubate for a period of 72 h to ensure effective knockdown. Subsequently, THP-1 cells underwent a 6-h pre-treatment with rhPCSK9. For cellular visualization, FITC labeling was achieved using a PKH67 green fluorescent cell linker kit (Sigma-Aldrich, PKH67GL). Then, a monolayer of human umbilical vein endothelial cells (HUVECs) was prepared for co-culture. FITC-labeled THP-1 cells were cocultured with the HUVECs for 1 h, both in the presence and absence of rhPCSK9. To remove non-adherent cells, two successive washes were conducted using cell culture media. Following this, we used a fluorescence microscope to capture detailed images of the THP-1 cells adhering to the HUVEC monolayer. Fluorescence-positive cells were quantified by counting using the ImageJ software (National Institutes of Health). Differences between means were analyzed using t-test with the Prism 6 software (GraphPad Software).
8
Exosome Uptake Imaging in HUVECs
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Exosomes were labeled using PKH67 (PKH67GL, Sigma-Aldrich) and incubated with HUVECs for 24 h.61 (link) Following incubation, the cells were fixed with 4% paraformaldehyde for 10 min, following which the cytoskeleton was stained using TRITC phalloidin (40734ES75, Yeasen Biotechnology, Shanghai, China), nuclei were stained using DAPI (ab104139, Abcam, Cambridge, MA, USA), and exosome uptake into HUVECs was visualized via confocal microscopy (FV10i, Olympus, Tokyo, Japan).
9
Exosome Purification and Internalization
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Exosomes were purified from CRC-derived conditioned medium (CM) by ultracentrifugation. CRC cell lines were cultured in DMEM supplemented with 3% exosome-depleted FBS (Exo-FBS-50A-1, SBI, Fremont, CA, USA). CM was collected after 48 h of cell culture and centrifuged at 500× g for 10 min at 4 °C, followed by centrifugation at 2000× g for 10 min at 4 °C. The supernatants were passed through a 0.22 µm filter (8020, IWAKI, Shizuoka, Japan) and ultracentrifuged at 174,900× g for 84 min at 4 °C. The exosomal pellets were washed with PBS, followed by a second ultracentrifugation at 174,900× g for 84 min at 4 °C, and then resuspended in PBS. An Optima WE-90 (Beckman, Brea, CA, USA) with SW32Ti as a swing rotor was used for ultracentrifugation. The amount of exosomes was measured as protein using the Qubit3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). When exosomes were exposed to cells, cell culture was performed by adjusting the medium so that the exosome concentration was 20 µg/mL.
To examine the cellular internalization of exosomes, exosomes were labeled with PKH67GL (Sigma-Aldrich, St. Louis, MO, USA), added to HUVECs at 80% confluence and incubated for 24 h before imaging under CLSM (FV3000, Olympus, Tokyo, Japan). The nucleus was labeled with Hoechst 33324.
To examine the cellular internalization of exosomes, exosomes were labeled with PKH67GL (Sigma-Aldrich, St. Louis, MO, USA), added to HUVECs at 80% confluence and incubated for 24 h before imaging under CLSM (FV3000, Olympus, Tokyo, Japan). The nucleus was labeled with Hoechst 33324.
10
Internalization of F-Hst Variants in HO1N1 Cells
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To study the internalization of the F-Hst and variants by HO1N1 cells 1 × 104 cells were distributed into 10 mm μ-Dishes (Ibidi GmbH, Munich, Germany) growth medium. After 24 h, the cells were washed with DPBS for 3 times. Thereafter, cells were incubated with NucBlue™ live cell stain following the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Cell membrane was labeled using PKH67GL (Sigma-Aldrich, St. Louis, MO, USA), following manufacturer’s instructions. Then serum-free DMEM/F12 containing the F-Hst variants at a final concentration of 2 μM. After 60 min of incubation with F-Hst variants, the cells were washed with DPBS. Subsequently, the cells were studied by a confocal laser scanning microscopy (CLSM) system (TCS SP8, Leica, Wetzlar, Germany) equipped with a 63 × 1.4 NA oil objective, and we chose the No. 20 Z-slice from the bottom (approximately 50 Z-slices per cell) since this slide contained the largest cross-section of nuclei.
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