Plasma-EVs, aged RBC-EVs, or EV subtypes were stained with PKH26 Red Fluorescent Cell Linker Dye (Sigma-Aldrich). EVs were washed twice with 10% exosome-free FBS in RPMI to quench the unbound dye. Recombinant annexin V (BD Biosciences) or functional grade blocking monoclonal antibodies against phosphatidylserine (PS) (Millipore), CD36 (Stemcell Technologies), CD163, CD206 (BioLegend), TLR1 (Invivogen), TLR2, and TLR4 (BioLegend) were used at multiple concentrations (0.01–2.0 µg/mL) to block EV–monocyte binding. In some experiments, EVs were incubated with annexin V or anti-PS antibody. In other experiments PBMCs were incubated with monoclonal antibodies against CD36, CD163, CD206, TLR1, TLR2, or TLR4 in a 5% CO2 incubator at 37°C for 1 h. PBMCs (500,000) were cultured with EVs, in a final volume of 0.5 mL for 24 h. PBMCs were stained with CD14-PerCP/Cy5.5 (BioLegend) and were fixed in 2% paraformaldehyde solution. PBMCs were subject to flow cytometry, and percent binding of monocytes to EVs was measured by gating on CD14+ cells.
Pkh26 red fluorescent cell linker dye
Manufactured by Merck Group
Sourced in United States
PKH26 is a red fluorescent cell linker dye manufactured by Merck Group. It is used to label and track cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanosep centrifugal device, by Pall Corporation (3 mentions)
Cd163, by BioLegend (1 mentions)
Recombinant annexin 5, by BD (1 mentions)
Cd14 percp cy5, by BioLegend (1 mentions)
Cd206, by BioLegend (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Nanosep, by Pall Corporation (1 mentions)
7 protocols using pkh26 red fluorescent cell linker dye
1
EV Binding to Monocytes: Receptor Blocking
2
Internalization of Labeled Exosomes
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For internalization assays, exosomes were isolated from BICR-18 cells (ExoB) or from AP-induced rats (ExoAP) and labelled with the PKH26 red fluorescent cell linker dye (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. The staining reaction was stopped with 3% bovine serum albumin (BSA) for 1 min. In order to remove the unbound dye, exosomes were washed three times with PBS using 300 kDa Nanosep centrifugal devices (Pall Corporation, New York, NY, USA). Fixed cells were also stained with the DNA-specific blue, fluorescent stain 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) for 1–5 min at room temperature. In some experiments, THP-1 macrophages were stained with the PKH67 green, fluorescent cell linker dye for general cell membrane.
3
Fluorescent Labeling of Exosomes and Cells
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For internalization assays, exosomes were labeled with the PKH26 red fluorescent cell linker dye (Sigma-Aldrich, St. Louis, MO) for 5 min. The staining reaction was stopped with 3% BSA for 1 min. In order to remove the unbound dye, exosomes were washed three times with PBS using 300 KDa Nanosep centrifugal devices (Pall Corporation, New York, NY). Cells were labeled with PKH67 green fluorescent cell linker dye, following the same protocol.
4
Cancer Cell-Neutrophil Adhesion Assay
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To evaluate attachment of cancer cells to neutrophils, neutrophils were initially seeded in 96-well plates (104 cells per well) and incubated for 4 h with cancer cell–conditioned media or with nonconditioned media. Where indicated, PMA (500 nM), CI-amidine (100 µM), GSK484 (10 µM), or DNase I (1 µg/ml) was added to the medium. Thereafter, neutrophils were gently washed, and fresh nonconditioned medium was added to all wells. Cancer cells that expressed GFP or that were labeled with PKH26 red fluorescent cell linker dye (Sigma-Aldrich) according to manufacturer’s instructions were then seeded onto neutrophils (2 × 104 cancer cells per well). At 1 h thereafter, wells were gently washed to remove unattached cancer cells. Attached cancer cells were viewed and counted under a fluorescence microscope. Where indicated, cultures were stained with Sytox Green dye and DAPI, as described for immunofluorescence staining studies. Three independent experiments were performed in which attached cancer cells were counted in four random ×200 microscopic fields in each experiment and where each experiment used neutrophils from a different donor.
5
Exosome Labeling and Uptake in HCECs
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To label the exosomes, we used the PKH26 red fluorescent cell linker dye (#MINI26, Sigma-Aldrich) following the manufacturer's instructions. The exosomes were stained by adding 1 μL of PKH26 dye to 200 μL of Diluent C fluid from the kit and incubating them for 5 min at room temperature (RT). To halt the labeling process, we added an equal volume of 10 % BSA and re-purified the exosomes using the total exosome isolation reagent precipitation method. The culture medium of HCECs was replaced with medium containing PKH26-labeled M1-derived exosomes. The cells were incubated with these exosomes for 16 h and then fixed with 4 % paraformaldehyde after washing with PBS. Subsequently, we labeled the cells with Wheat Germ Agglutinin (WGA; #W11261, Invitrogen) for 30 min at RT and stained the nuclei with 4′,6-diamidino-2-phenylindole (DAPI). The cells were analyzed using an inverted fluorescence microscope (DMi8, Leica, Wetzlar, Germany).
6
EV Labeling with PKH26 Dye
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For internalization and binding assays, EVs were labeled with the PKH26 red fluorescent cell linker dye (Sigma Aldrich) for 5 min. The staining reaction was stopped with 3% BSA for 1 min. EVs were washed three times with PBS in order to remove the unbound dye, using 300 KDa Nanosep centrifugal devices (Pall Corporation).
7
Exosome Biodistribution Tracking
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For tracking experiments, exosomes were labeled with PKH26 Red Fluorescent Cell Linker Dye (Sigma, St Louis, MO) according to the supplier’s specifications. The staining reaction was stopped with 3% BSA for 1 min, and the labeled exosomes (Exo-PKH26) were washed three times with PBS in order to remove the unbound dye, using 300 KDa Nanosep centrifugal devices (Pall Corporation). For each group, PBS stained with PKH26 was used as a control.
In a first tracking analysis, 7 µg of Exo-PKH26 obtained from Control or AP plasma samples were resuspended in 1 ml of saline solution and perfused through the inferior vena cava of control animals at a rate of 6 ml/h during 10 min as previously described9 (link). After 30 min, animals were sacrificed and samples of pancreas, liver, lung, kidney and small intestine were obtained and processed for the histological analysis.
In a second experiment, Exo-PKH26 from PAAF samples were perfused to control animals through the hepatic portal vein at a rate of 4 ml/h and livers from portal-perfused animals were obtained for immunofluorescence analysis.
In a first tracking analysis, 7 µg of Exo-PKH26 obtained from Control or AP plasma samples were resuspended in 1 ml of saline solution and perfused through the inferior vena cava of control animals at a rate of 6 ml/h during 10 min as previously described9 (link). After 30 min, animals were sacrificed and samples of pancreas, liver, lung, kidney and small intestine were obtained and processed for the histological analysis.
In a second experiment, Exo-PKH26 from PAAF samples were perfused to control animals through the hepatic portal vein at a rate of 4 ml/h and livers from portal-perfused animals were obtained for immunofluorescence analysis.
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