SH-SY5Y cells (a gift from Dr. R. de Silva, University College London Institute of Neurology [UCL ION]) were seeded at 4 × 105 cells/well in 6-well plates in DMEM with 10% heat-inactivated bovine calf serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). The following day, cells were loaded with Vybrant CM-Dil dye (1:200; Thermo Fisher Scientific) for 15 min, and the medium was changed to 500 μL PBS. Cells were irradiated with 500 J/m2 using a UV Crosslinker (Stratech, UK) and then incubated for 24 hr in normal DMEM. Apoptosis was confirmed using fluorescein isothiocyanate (FITC)-labeled annexin V (Miltenyi; see Figure S4 B). Dye-labeled apoptotic SH-SY5Y cells were harvested in PBS without Ca2+/Mg2+, spun down, resuspended in macrophage end-differentiation medium, and counted. A total of 500,000 apoptotic SH-SY5Y cells were added to each well of the iPSC-MGLCs (seeded at 1 × 105 cells/well in 24-well plates for 7–9 days and changed into 200 μL of fresh macrophage end-differentiation medium before the assay) for 2 hr. As a negative control, iPSC-MGLCs were pre-incubated for 30 min with 10 μM cytochalasin D (Sigma). iPSC-MGLCs were harvested with trypsin LE (Life Technologies) before being resuspended in FACS buffer and analyzed using a Becton Dickinson FACSCalibur analyzer. Data were analyzed using Flowing software version 2.5.1.
Vybrant cm dil dye
Manufactured by Thermo Fisher Scientific
Vybrant CM-Dil dye is a fluorescent cell labeling reagent used for the long-term tracking and monitoring of viable cells. It is a lipophilic and cationic dye that can passively diffuse across the cell membrane and stain viable cells. The dye exhibits red-orange fluorescence when excited by light.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur analyzer, by BD (1 mentions)
Trypsin le, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Akta start, by GE Healthcare (1 mentions)
Capto core 700 hitrapcolumns, by GE Healthcare (1 mentions)
2 protocols using vybrant cm dil dye
1
Phagocytosis of Apoptotic Neuroblastoma Cells by iPSC-Derived Microglia-Like Cells
2
Drug Loading and Purification of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
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A
concentration of 5 × 1012 EVs (either total EVs or
CD81 + AP EVs) was diluted into 450 μL of fresh 1× PBS.
Dimethyl sulfoxide (DMSO) was added to the EVs to a final concentration
of 5% to facilitate drug solubility. Drugs were then incubated with
purified EVs at 4 °C for 24 h with continuous rocking. Doxorubicin
and paclitaxel Oregon Green 488 (Flutax-2, Invitrogen P22310) were
added to a final concentration of 64 ng/μL. For non-specific
membrane labels, the Vybrant CM Dil dye (ThermoFisher V22888) was
added to a final concentration of 10 μM. After incubation, the
EV mixtures were then loaded onto equilibrated Capto Core 700 HiTrap
columns (GE Healthcare 17-5481-51). Fractions were collected using
the AKTA Start (GE Healthcare 29022094-ECOMINSSW) into sterile 1.5
mL tubes. The settings used for fraction collection are outlined inTable S2 .
Drug or membrane fluorophore
(DiI or CellMask) EV fractions were identified using the BMG LabTech
FluorStar Optima plate reader. Fractions were further validated for
the presence of EVs using nanoparticle tracking analysis (NTA—see
below). Fractions of high concentrations of EVs and fluorescence were
pooled together.
concentration of 5 × 1012 EVs (either total EVs or
CD81 + AP EVs) was diluted into 450 μL of fresh 1× PBS.
Dimethyl sulfoxide (DMSO) was added to the EVs to a final concentration
of 5% to facilitate drug solubility. Drugs were then incubated with
purified EVs at 4 °C for 24 h with continuous rocking. Doxorubicin
and paclitaxel Oregon Green 488 (Flutax-2, Invitrogen P22310) were
added to a final concentration of 64 ng/μL. For non-specific
membrane labels, the Vybrant CM Dil dye (ThermoFisher V22888) was
added to a final concentration of 10 μM. After incubation, the
EV mixtures were then loaded onto equilibrated Capto Core 700 HiTrap
columns (GE Healthcare 17-5481-51). Fractions were collected using
the AKTA Start (GE Healthcare 29022094-ECOMINSSW) into sterile 1.5
mL tubes. The settings used for fraction collection are outlined in
Drug or membrane fluorophore
(DiI or CellMask) EV fractions were identified using the BMG LabTech
FluorStar Optima plate reader. Fractions were further validated for
the presence of EVs using nanoparticle tracking analysis (NTA—see
below). Fractions of high concentrations of EVs and fluorescence were
pooled together.
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