HUVECs cultured with and without pNPs and BA NPs (20 μM) were imaged using a Zeiss LSM 710 CLSM (Jena, Germany). The HUVECs were seeded in complete DMEM in a humidified atmosphere with 5% CO2 and then cultured at 37°C overnight. Then, 1 ml of serum-free fresh medium containing pNPs (20 μM) was used for replacing the medium, and the cells were cultured for 1, 2, and 4 hours and washed with PBS three times before being imaged using a Zeiss LSM 710 CLSM with a 63× objective lens. The MCF-7 cells and HUVECs cultured with pNPs and BA NPs were imaged in the same way.
Lsm 710 clsm
Manufactured by Zeiss
Sourced in Germany
The LSM 710 is a confocal laser scanning microscope (CLSM) manufactured by Zeiss. It is designed for high-resolution imaging of fluorescent samples. The LSM 710 uses a focused laser beam to excite fluorophores within the sample, and the emitted fluorescence is detected and reconstructed into a digital image.
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Lab products found in correlation
Lsm 780 clsm, by Zeiss (2 mentions)
Imaris software package, by Oxford Instruments (2 mentions)
Eos 550d camera, by Canon (1 mentions)
Leica dm il led inverted microscope, by Leica Microsystems (1 mentions)
Plan apochromat 63 1.4 objective, by Zeiss (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Lsm880airyscan clsm, by Zeiss (1 mentions)
Plan apochromat 3 10x 0.45 m27 air objective, by Zeiss (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Live dead baclight bacterial viability kit for microscopy, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Calibur flow cytometry, by BD (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Hmga2 antibody, by Cell Signaling Technology (1 mentions)
Cd31 antibody, by Agilent Technologies (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fls980 fluorometer, by Edinburgh Instruments (1 mentions)
20 protocols using lsm 710 clsm
1
HUVEC Imaging with Nanoparticles
2
Microscopic Imaging of Cell Morphology
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Phase contrast microscopy was performed before and after the RPM experiments, to ensure viability and determine the morphological changes of the cells. Pictures were taken using a Canon EOS 550D camera (Canon GmbH, Krefeld, Germany) through a Leica DM IL LED inverted microscope (Leica Microsystems, Wetzlar, Germany). Fluorescence staining was analysed using a Zeiss LSM 710 CLSM (Zeiss, Jena, Germany) fitted with a Plan-Apochromat 63 × 1.4 objective, as previously described [26 (link),85 (link)]. Excitation and emission wavelengths for the Alexa Fluor 488 were λex = 488 nm and λem = 525 nm, respectively. Correspondingly, for the TRITC: λex = 532 nm and λem = 576 nm.
3
Intracellular Trafficking of RGP Nanoparticles
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The HeLa cells were inoculated in a 6-well plate with a sterilized coverslip at an initial density of 2.5×105 cells/well and cultured at 37°C overnight. Subsequently, the cells were incubated with 1 mL FBS-free DMEM containing RGP nanoparticles (RNase A concentration of 4 μg/mL) for 2 and 6 hrs, respectively. Afterward, the cells were stained with LysoTracker Green DND-26 (ThermoFisher, Eugene, OR) for 5 mins as described in previous reports.31 (link)–33 (link) After washing with PBS three times, the cells were fixed with 4% paraformaldehyde solution for 15 mins and stained with DAPI solution (1 μg/mL) for 5 mins. Finally, the coverslip was subjected to the analysis on an LSM 710 CLSM (Carl Zeiss Microscopy LLC to detect the endosomal escape of RGP nanoparticles.
4
Confocal Imaging of Confetti Proteins
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Confocal microscopy was performed using Zeiss LSM710 CLSM, Zeiss LSM780 CLSM and Zeiss LSM880Airyscan CLSM instruments. The settings for the imaging of Confetti fluorescent proteins were previously described (Snippert et al., 2010 (link)). The imaging of the confocal stack was done with a Zeiss LSM780 CLSM, Plan-Apochromat 3 10x/0.45 M27 Zeiss air objective.
5
Nanoflower Internalization in Tumor Spheroids
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Multicellular tumor spheroid (Friedrich et al., 2009) was rendered for testing the internalization of nanoflower. The K562/vcr cells were seeded and cultured overnight. The T75 flask was pre-covered by 10 mL of hot agarose (1 w/v %) and cooled to completely solidified. 106 cells were seeded in a flask and incubated for 72 h. The MCSs were treated with NF-PV (without aptamer) or KNf-PV (with aptamer). The drug concentration was 100 μg/mL based on VCR. After 4 h of incubation at 37°C, the spheroids were collected, washed with PBS for three times, and stained by Calcein-AM for 1 h at room temperature. The spheroids were fixed with PFA 4% (w/v) in PBS for 1 h at room temperature and observed with confocal laser scanning microscopy (LSM 710 CLSM, Carl Zeiss, Jena, Germany).
6
Visualization of sPG Cellular Localization
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ATDC5 or NHDF cells were incubated in the presence of ATTO 488-labeled sPG or ATTO 488 for 30 min. The cells were washed with PBS, then added with CellMask Deep Red (Thermo Fisher Scientific, Waltham, MA, USA) to recognize cell membrane and incubated for 30 min. The cells were also washed with PBS, followed by Live Cell Imaging with a LSM 710 CLSM (Carl Zeiss, Oberkochen, Germany) equipped with Temp Module and CO2 Module. The obtained optical sectioning (z-stack) was subjected to three-dimensional reconstruction using ZEN (Carl Zeiss, Oberkochen, Germany).
7
Viability Assay of E. faecalis V583
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Viability of E. faecalis V583 treated or not with SP was analyzed using the LIVE/DEAD BacLight Bacterial Viability Kit for microscopy (L7007, Invitrogen). In this system, live bacteria stain with SYTO 9 to produce a green fluorescence whereas dead bacteria stain with propidium iodide (PI) to produce a red fluorescence. Briefly, 3 µl of the SYTO 9/PI mixture, prepared according to the manufacturer’s instruction, was added to the bacterial cells previously treated with 10−2, 10−4 or 10−6 M of SP for 2 h, and untreated cells were kept as control. The samples were incubated for 15 min in dark at room temperature and 5 µl of this sample was trapped in between coverslip and glass slide. The slide was viewed under a confocal laser-scanning microscope (LSM 710 CLSM, Zeiss), using 60× objective sequentially using fluorescence setting for FITC (green/SYTO 9, viable cells) and PI (red/PI, dead cells) filters, respectively, followed by phase contrast and bright field settings. SYTO 9 and PI images were merged and acquired using ZEN® software (Zeiss).
8
Ru-1 Induced PML Localization
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NB4 cells cultured in 24-well plates at a density of 100 × 104 cells per well were treated with 30 μM Ru-1 for 4, 8, 12, 16, 20 or 24 hours. After then, the cells were harvested and pelleted onto slides. The slides were quickly air-dried and the cells were immobilized using 4% paraformaldehyde (PFA). After permeabilization and blocking treatment, immunofluorescence staining of the PML was performed using PML antibodies and AF594 labeled secondary antibodies. All slides were examined under a LSM 710 CLSM (Carl Zeiss, Jena, Germany).
9
RGP Transfection and Cell Analysis
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The HeLa cells were obtained from the Shanghai Institute of Cell Bank (Shanghai, China) and inoculated in 6-well plates at a density of 3.0×105 cells/well. After the culture at 37°C overnight prior to the transfection, the medium was removed, and the cells were incubated with RGP at different concentration (0.5–8.0 μg/mL RNase A) in FBS-free DMEM for 4 hrs. Then, the medium was discarded, and the cells were collected, suspended in 1 mL PBS and analyzed through Calibur flow cytometry (BD Bioscience, Mountain View, USA) with excitation and emission wavelengths of 488 and 575 nm, respectively. For the confocal laser scanning microscope (CLSM) analysis, HeLa cells were seeded into 6-well plates with sterilized coverslips at a density of 2.2×105 cells/well and cultured at 37°C overnight. Subsequently, 2 mL of FBS-free DMEM was added into each well after removing the medium, and the cells were transfected with 4 μg/mL of RGP solution for 6 hrs. The medium was discarded after the transfection, and the cells were washed with PBS three times, fixed with 4% paraformaldehyde solution for 15 mins and stained with DAPI solution (1 μg/mL) for 5 mins. Finally, the coverslips were observed on an LSM 710 CLSM (Carl Zeiss Microscopy LLC, Jena, Germany).
10
Biofilm Microscopic Analysis Protocol
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