PLC/PRF/5 cells were plated in an 8-well chamber at 3000 cells/well. After treatment of cells with the formulations of SRFT and Pull-SA SRFT at 8 µM concentration after sonication for 10 min, the cells were incubated for 48 h. DNA nuclear morphology was examined with Hoechst dye (0.1µg/mL) staining for 10 min. The cells were washed once with PBS and visualized and analyzed using Leica SP 8 WLL Confocal Laser Scanning Microscope.
Sp 8 wll confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany
The SP 8 WLL Confocal Laser Scanning Microscope is a high-performance imaging system designed for advanced microscopy applications. It features a white-light laser source that provides a wide range of excitation wavelengths, enabling the visualization of a variety of fluorescent samples. The system's confocal technology allows for optical sectioning, allowing users to capture detailed, high-resolution images with improved contrast and reduced background noise.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Oct compound, by Thermo Fisher Scientific (2 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Evos fl auto 2 imaging system, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Incucyte system, by Sartorius (1 mentions)
7 protocols using sp 8 wll confocal laser scanning microscope
1
Examining SRFT and Pull-SA SRFT Cytotoxicity
2
Apoptosis Evaluation by FACS and CLSM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect early apoptosis, cells were seeded in a 6-well plate at a cell density of 1×106 cells/well for FACS analysis and also in an 8-well chamber at a density of 3×103 for CLSM imaging and incubated overnight. The next day, cells were treated with formulations of SRFT and Pull-SA SRFT at 8 µM concentration after sonication for 10 min. Untreated cells served as the control group. After incubation for 12 h, the cells were trypsinised and stained with Annexin V-FITC and propidium iodide (PI) in the dark for FACS analysis39 (link) and the cells were sorted using BD FACS AriaTM III Cell Sorter, USA, and analyzed using BD FACSDiva 7.0 software. In addition, the cells treated for the imaging study were stained according to the kit protocol and visualised using Leica SP 8 WLL Confocal Laser Scanning Microscope (CLSM).
3
Immunostaining of hiPSC-Derived Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HiPSC-CMs were fixed with 4% PFA in PBS for 10 min and permeabilised with PBS/0.1% Triton-X-100 for 7 min at room temperature. The cells were then blocked in blocking solution containing PBS/1% BSA for 1 h at room temperature and incubated overnight at 4 °C with anti-alpha sarcomeric actinin (ACTN2 mouse monoclonal, Sigma‐Aldrich #A7811, dilution 1:1000) and anti-TFEB (rabbit polyclonal, Cell Signaling #4240S, dilution 1:200). After washing, hiPSC-CMs were incubated for 1 h at room temperature in the dark with the appropriate fluorochrome-conjugated secondary antibody Donkey anti-Rabbit Alexa Fluor 488 (1:300) and Donkey-anti-mouse AF594 (1:300). Nuclei were counterstained with DAPI (all Thermo Fisher Scientific). Images were acquired with a Leica SP8 WLL confocal laser-scanning microscope using ×63 magnification objective and Z stack acquisition.
4
Visualizing Nanoparticle Uptake in HCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To visualise the ability of HCC cells to uptake Pull-SA nanoparticles, cells were seeded in an 8-well chamber at a density of 3×103 cells/well followed by incubation for 24 h for cells to attach. Then, coumarin-6-loaded Pull-SA nanoparticles formulated by the dialysis method, were added to cells (at a concentration of 250 ng/mL)38 (link) after sonication for 10 min and incubated for 4 h. After incubation, the cells were carefully rinsed twice with PBS to remove any unbound particles. The plasma membrane was stained red with Cell MaskTM (plasma membrane staining kit) for 10 min and the nucleus was counterstained blue with Hoechst for another 10 min. The cells were then analysed using Leica SP 8 WLL Confocal Laser Scanning Microscope (CLSM).
5
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized for 10 minutes with PBS containing 0.1% Triton X-100 (Sigma-Aldrich), and blocked for 1 h with PBS containing 5% BSA (Sigma-Aldrich). Then cells were stained with primary antibody overnight at 4 °C. The next day, cells were washed 3 times (20 minutes each time) with PBS. After that, cells were incubated with fluorochrome-conjugated secondary antibodies for 1 h at room temperature and washed 3 times (20 minutes each time) with PBS. Then, cells were stained with DAPI (Life Technologies) for 10 minutes at room temperature and washed once with PBS for 10 minutes. Both primary and secondary antibodies were diluted in 5% BSA/PBS. Images were taken with the EVOS FL AUTO2 imaging system (Thermo Fischer Scientific) with a 20× objective or using the Incucyte system (Sartorius). Confocal imaging was done using a Leica SP8WLL confocal laser-scanning microscope using a 63× objective and z-stack acquisition. Details of all antibodies used are provided in Supplementary Table S1 .
6
Quantification of Tumor Cell Metastasis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In total, 2 × 106 (in 100 μL of PBS per mouse) AF9-depleted MCF-7 cells with or without reconstituted expression of rAF9 WT or Vehicle were injected into randomized 6-week-old female athymic nude mice per group via the lateral tail veins. The mice were sacrificed after 3 h for the quantification of the total tumor cells (including intravascular and extravascular tumor cells) in lung tissues or 48 h for the quantification of extravascular tumor cells in lung tissues. The lungs were fixed in 4% formaldehyde and embedded in optimum cutting temperature (OCT) compound (Thermo) after dehydration by 30% sucrose solution. The lung tissues were systematically sectioned through the entire lung with one 50 μm section obtained in every 0.2 mm of lung thickness. The tissue sections were washed with PBS and 0.3% Triton X-100 and blocked in PBS with 10% normal goat serum, followed by incubation with the primary antibody. After washing, the samples were incubated with corresponding secondary antibodies and DAPI. Confocal images were obtained using a Leica SP8 WLL confocal laser scanning microscope (Leica, Germany) and ImageJ software.56 (link),57 (link)
7
Cancer Cell Metastasis Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2 × 10 6 (in 100 μl of PBS per mouse) AF9-depleted 786-0 cells with or without reconstituted expression of rAF9 WT or Vehicle were injected into randomized 6-week-old female athymic nude mice per group via lateral tail veins. The mice were sacrificed after 3 h for counting total tumour cells (including intravascular and extravascular tumour cells) in lung tissues or 48 h for counting extravascular tumour cells in lung tissues. The lungs were fixed by 4% formaldehyde, and were embedded in optimum cutting temperature (OCT) compound (Thermo) after dehydration by 30% sucrose solution. Lung tissues were systematically sectioned through the entire lung with one 50 μm section taken in every 0.2 mm of lung thickness. The tissue sections were washed in PBS, 0.3% triton X-100 and blocked in PBS with 10% normal goat serum followed by the primary antibody incubation. After washing, samples were incubated with corresponding secondary antibodies and DAPI. Confocal images were obtained using a Leica SP8 WLL confocal laser scanning microscope (Leica, Germany) and ImageJ software [50, (link)51] (link).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!