HEK-293 cells were plated on poly-d -lysine coated chambers (ibidi, Germany) in DMEM supplemented with 10% (vol/vol) FBS (DMEM/FBS). After 24 h, cells were transfected with 300 ng of rat (r) NK1R-GFP per chamber and cultured for 48 h. To identify endosomal compartments, HEK-293 cells were infected with Rab5a-GFP (resident in early endosomes) or Rab7a-GFP (late endosomes) CellLight BacMam2.0 (Thermo Fisher Scientific) for 16 h. To examine localization of nanoparticles, cells were incubated in Leibovitzś L-15 medium with DIPMA-Cy5 nanoparticles (20 μg ml−1, 30 min, 37 °C) or vehicle, followed by addition of SP (10 nM). Cells were imaged at 30 and 60 min post-SP addition using a Leica SP8 confocal microscope equipped with HCX PL APO ×40 (NA 1.30) and HCX PL APO ×63 (NA 1.40) oil objectives. Images were analysed using Fiji36 (link) and deconvolved with Huygens Professional version 18.04 (Scientific Volume Imaging, http://svi.nl ), using the CMLE algorithm with a signal-to-noise ratio of 10 and 100 iterations. Co-localization was evaluated by determination of the Manders overlap coefficient25 .
Celllight bacmam 2
Manufactured by Thermo Fisher Scientific
CellLight BacMam 2.0 is a live-cell labeling reagent that enables the visualization of cellular structures and organelles in a wide range of cell types. It uses baculovirus technology to efficiently deliver fluorescent protein-based constructs into cells, allowing real-time imaging of various cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Hcx pl apo 40, by Leica (1 mentions)
Huygens professional version 18, by Scientific Volume Imaging (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Nis element, by Nikon (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Latrunculin b, by Bio-Techne (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Staurosporine, by LC Laboratories (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
5 protocols using celllight bacmam 2
1
Nanoparticle Trafficking in Transfected HEK-293 Cells
2
Tracking Glioblastoma Cell Interactions
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U87EGFRwt nuclei were stained with Hoechst 33342, (SC-495790) or labeled with NLS GFP-Luciferase virions (CellLight™ BacMam 2.0, Thermo Fisher). 24 wells plates were coated overnight at 4 °C with 5 μg/ml laminin (LaminStem™ 521, BI #05-753-1F, Biological Industries). After washing, 2 × 104 stained cells were co-seeded with unstained 2 × 103 U87EGFRvIII cells, and tracked for 18 h. In some experiments 1.4 × 104 stained U87EGFRwt cells were cultured in 9 well Ibidi culture plate for 24 h under conditioned medium (CM) of U87EGFRvIII cells (CM; collected following 72 h of incubation with cell in mild starvation(1% FCS)). At each time point multipoint snapshots of ten fields (~150 cell/field) were imaged using 10 × /0.3 Plan Fluor lens mounted on an Eclipse Ti Nikon microscope. Image sequences using Zyla sCMOS (ANDOR) camera, were taken every 20 min. Coordinates of the centers of each labeled nuclei, trajectory, velocity and other parameters were obtained for each cell, in each frame using the Nikon software (NIS-elements).
3
Visualizing Lysosome and Golgi in MCF-7 Cells
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MCF-7 cells are seeded out at a density of (10 000–15 000) cells per well in an 8 well μ-slide (no. 1.5 polymer coverslip, tissue culture treated, Ibidi™) 48 hours prior to imaging. CNDs are added to respective wells to yield a concentration of 500 μg mL−1. The cells in the respective chambers were transfected with reagents from the CellLight™ BacMam 2.0 product series (Invitrogen™) 24 hours prior to imaging. To label the lysosomes CellLight™ Lysosomes-RFP and CellLight™ Lysosomes-GFP was used yielding MCF-7 cells expressing a fusion protein of LAMP1 and the respective fluorescent protein. To label the Golgi apparatus CellLight™ Golgi-GFP was used yielding MCF-7 cells expressing a fusion protein of the human Golgi resident enzyme N-acetylgalactosaminyltransferase and GFP. One hour before imaging the samples were washed with PBS and the medium was exchanged with fresh full medium. Between the preparation steps the sample was kept in the incubator at 37 °C and 5% CO2.
4
Quantifying NP Subcellular Localization in HT1080 Cells
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To quantify
subcellular localization of NPs in HT1080 cells, Rab7a-RFP and Lamp1-RFP
fusion constructs were expressed using a commercial baculovirus platform
(CellLight BacMam 2.0, Invitrogen), following manufacturing guidelines.
Pharmacological modulation of NP uptake was performed using the following:
staurosporine (1 μM; LC laboratories), latrunculin B (1 μM;
Tocris), cytochalasin D (1 μM; Sigma), chloroquine (50 μM;
Sigma), and ethylisopropyl amiloride EIPA (100 μM; Tocris).
Cells were rinsed in fresh media immediately prior to imaging; only
adherent cells were quantified. chloroquine dose–response measurements
were normalized to the C16proDOX control (rather than the
chloroquine-free control), in order to compare relative effects on
the potency of Pd-mediated C16proDOX activation itself.
50 μM chloroquine alone caused a 15–30% decrease in cell
viability.
subcellular localization of NPs in HT1080 cells, Rab7a-RFP and Lamp1-RFP
fusion constructs were expressed using a commercial baculovirus platform
(CellLight BacMam 2.0, Invitrogen), following manufacturing guidelines.
Pharmacological modulation of NP uptake was performed using the following:
staurosporine (1 μM; LC laboratories), latrunculin B (1 μM;
Tocris), cytochalasin D (1 μM; Sigma), chloroquine (50 μM;
Sigma), and ethylisopropyl amiloride EIPA (100 μM; Tocris).
Cells were rinsed in fresh media immediately prior to imaging; only
adherent cells were quantified. chloroquine dose–response measurements
were normalized to the C16proDOX control (rather than the
chloroquine-free control), in order to compare relative effects on
the potency of Pd-mediated C16proDOX activation itself.
50 μM chloroquine alone caused a 15–30% decrease in cell
viability.
5
Multicolor Organelle Labeling Protocol
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Commercially available dyes DAPI, LysoTracker Deep Red (Invitrogen), Rab7a-GFP (CellLight BacMam 2.0, Invitrogen) and MitoTracker Deep Red were used in co-localisation studies for the determination of the localisation of RuBDP NPs following cell uptake. The nuclear staining DAPI dye was added at 3 μM concentration and incubated for 20 min prior to imaging. DAPI was excited using a 405 nm laser and the emission was collected between 425 and 580 nm. LysoTracker Deep Red, used for staining of lysosomes, was added at 50 nM and incubated for 75 min prior to imaging (λexc 647 nm, λem range: 650–800 nm). Rab7a-GFP, used to stain late endosomes, was added to the cell dish and incubated overnight at 37 °C prior to the addition of RuBDP NPs. Rab7a-GFP was excited using a 488 nm white light laser and the emission was collected between 490 and 540 nm. MitoTracker Deep Red (25 nM) was incubated for 40 min prior to imaging and was excited at 644 nm with emissions collected between 650 and 800 nm. Following incubation, the dye/growth medium was removed, and the cells were washed with supplemented PBS prior to imaging. The fluorescence intensity profiles were obtained using ImageJ.
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