RCC cells were cultured in 4-chamber slides as described above. At approximately 80% confluence, the monolayer cell culture was rinsed briefly in PBS. Next, the cells were covered in 4% paraformaldehyde (PFA) for 10 min at room temperature. The PFA was removed by washing with PBS (3 times) and goat serum (10%) in PBS was used for blocking for 1 h at room temperature. The cells were incubated separately with diluted primary antibodies against CB1 (1:1000) and CB2 (1:1000) at 4 °C for 4 h. The cells were washed three times with PBS and then incubated with Alexa Fluor® 546 secondary goat anti-rabbit antibody (1:400) for 2 h at room temperature away from light. Again, the cells were rinsed 3 times with PBS, followed by incubation with DAPI (1:5000) (ThermoFisher Scientific, Massachusetts, USA) for 10 min. For the control, the cells were incubated only with secondary antibody. The slides were washed with PBS and covered with coverslips using CoverGrip Sealant (Biotium, California, USA), and images were captured using an Olympus CKX41 fluorescence microscope.
Ckx41 fluorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The CKX41 is a fluorescence microscope designed for basic fluorescence imaging. It features high-intensity LED illumination for fluorophore excitation and a sensitive monochrome camera for fluorescence detection. The CKX41 is capable of capturing fluorescence images but a detailed product description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Covergrip sealant, by Biotium (2 mentions)
Oxiselect comet assay kit, by Cell Biolabs (1 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Celltiter glo ctg, by Promega (1 mentions)
R19 15, by BD (1 mentions)
Prism software, by GraphPad (1 mentions)
Digital video camera, by Sony (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Dp73 digital camera, by Olympus (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Rhodamine conjugated phallotoxin, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
20 protocols using ckx41 fluorescence microscope
1
Immunofluorescence Analysis of CB1 and CB2
2
RCC Sphere Immunophenotyping Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RCC cells were counted and seeded at density of 100 cells/well in ultra-low attachment 24 wells plates (TC plate, suspension, F, Sarstetd, Numbrecht, Germany) supplemented with sphere promoting media as described previously20 (link),77 (link). Later culture media was removed, and tumor spheres were rinsed briefly in PBS (3 times). 10% goat serum in PBS was used for blocking at room temperature (1 hr). Tumor spheres were incubated separately with diluted primary antibodies against CD105 (1:1000), CD133 (1:500), CD44 (1:1000) and CXCR4 (1:1000) at 4 °C for 4 h; washed three times with PBS and incubated with Alexa Fluor 488 secondary goat anti-mouse antibody (1:400) for 1 h at room temperature. The spheres were rinsed 3 times with PBS, followed by incubation with DAPI (1:5000; ThermoFisher Scientific, Massachusetts, USA) for 10 min. As control the spheres were incubated only with secondary antibody. The slides were washed with PBS and covered with coverslips using CoverGrip Sealant (Biotium, California, USA), and images were captured using an Olympus CKX41 fluorescence microscope.
3
Comet Assay for DNA Damage Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A DNA damage assay was carried out using an OxiSelect Comet Assay kit (STA-351, Cell Biolabs, San Diego, CA, USA). Briefly, cells were seeded in a 6-well plate, treated with or without BP (MDA-MB-231: 50 μg/mL, MCF-7: 75 μg/mL) for 24 h, and then exposed to 6 Gy IR. After 48 h, the cells were harvested and the comet assay performed according to the manufacturer’s instructions. The comet tails were imaged using an Olympus CKX41 fluorescence microscope at ×400 magnification. The images were analyzed using OpenComet software (v1.3.1, OpenComet is an open-source software tool providing automated analysis of comet assay images. The OpenComet plug-in and source code is available at www.opencomet.org .) [26 (link)].
4
Analyzing Membrane Tethers in Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PMA4/PAAm4 coated microfluidic slides, PMA4/PAAm4 coated microfluidic slides with DOTAP, or a low attach 24-well plate (50,000 cells/channel). Cells were incubated for 1 hr to allow for tethering. After 1 hr, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to the top port on the DOTAP slides. This wash was to ensure only tethered cells were analyzed. After this wash, CellMask Orange (Life Technologies) cell membrane dye was added to each channel to a final concentration of 1:10,000. McTNs were scored blindly in a population of 100 cells/well as previously described [12 (link)]. Representative images were taken at 40x magnification with an Olympus CKX41 fluorescence microscope.
5
Immunofluorescence Staining of IgG1-Producing Cells
6
Evaluating Co-Culture Media Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The co-culture media at 1:1 ratio of target cell (NPC43, HK1, C666–1, C17, B110, and G517) media and effector cell (NK-92) media was evaluated for any deleterious effect on the growth of target cells during co-culture. The target cells were first seeded into a 24- or 96-well plate in their own respective culture medium until cell attachment. Cell viability of the target cells in individual culture medium and in co-culture medium at 0 and 72 h was validated by using CellTiter-Glo (CTG; Promega, USA, #G7571), an established cell viability assay. Cell morphology was also observed by using Olympus CKX41 fluorescence microscope and bright field images were acquired at 24, 48 and 72 h.
7
Alzheimer's Mouse Model Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fourteen animals B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/Mmjax mice aged 1 year and 7 months were used (Alzheimer´s model). The mice were anesthetized with a mixture of ketamine–xylazine–acepromazine (4:4:1), perfused with filtered PBS (pH 7.2), and fixed by perfusion of approximately 50 mL of parafolmaldehyde 4% in PBS. Then, the mice brains were removed.
Subsequently, the brains were left in fixation solution overnight (PFA 4% in PBS pH 7.2) and transferred to a maintenance solution (sucrose 30%, sodium azide 0.2% in PBS pH 7.2). Finally, the brains were cut in a cryostat (30 μm slices), obtaining a total of 41 slices which were mounted on the slides. The AD mice brain slides were incubated with CRANAD-2 0.24 mM for 5 min (200 μL of solution was added to each tissue slice). Then, the excess of dye was removed and the tissue washed with 50% ethanol, followed by absolute ethanol. Finally, the slides were incubated for 5 min with different concentrations of GNR-PEG-D1 in order to determine the optimal concentration needed. We evaluated five concentrations: 0.5, 0.1, 0.01, 0.001, and 0.0001 nM (200 μL of solution was added to the tissue). Imaging was performed with an Olympus CKX41 fluorescence microscope with a 10x objective before and after each incubation step. The fluorescence intensity was evaluated both in the presence and in the absence of GNRs in the same histological section.
Subsequently, the brains were left in fixation solution overnight (PFA 4% in PBS pH 7.2) and transferred to a maintenance solution (sucrose 30%, sodium azide 0.2% in PBS pH 7.2). Finally, the brains were cut in a cryostat (30 μm slices), obtaining a total of 41 slices which were mounted on the slides. The AD mice brain slides were incubated with CRANAD-2 0.24 mM for 5 min (200 μL of solution was added to each tissue slice). Then, the excess of dye was removed and the tissue washed with 50% ethanol, followed by absolute ethanol. Finally, the slides were incubated for 5 min with different concentrations of GNR-PEG-D1 in order to determine the optimal concentration needed. We evaluated five concentrations: 0.5, 0.1, 0.01, 0.001, and 0.0001 nM (200 μL of solution was added to the tissue). Imaging was performed with an Olympus CKX41 fluorescence microscope with a 10x objective before and after each incubation step. The fluorescence intensity was evaluated both in the presence and in the absence of GNRs in the same histological section.
8
Anti-nuclear Autoantibody Quantification in MRL/Fas Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MRL/Faslpr/lpr mice were started on HDI-water ad libitum at 6- or 17-weeks of age, or were on untreated water throughout their life and scarified when moribund. Anti-nuclear antibody (ANA) and anti-dsDNA antibody titers were determined in sera. For ANA assays, sera were serially diluted in PBS (from 1:40 to 1:160), incubated on antinuclear Ab substrate slides (HEp-2 cell-coated slides, MBL-BION) and detected with a 1:1 mixture of FITC-anti-IgG1 and FITC-anti-IgG2a mAbs (R19-15; BD Biosciences). Images were acquired with a 40× objective on an Olympus CKX41 fluorescence microscope. Anti-dsDNA IgG and IgG2a antibody titers were measured in sera of MRL/Faslpr/lpr mice by ELISA as previously described (39 (link)). Titers were expressed in relative units (RUs), defined as the dilution factor needed to reach 50% of binding saturation and calculated using Prism? software (GraphPad Software, Inc.).
9
Immunofluorescence Analysis of Spi-C Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMMs were seeded into 96-well plates at a density of 3 × 104 cells/well. After incubation with RANKL (200 ng/ml) and/or inhibitors, the cells were fixed with 4% paraformaldehyde for 5 min. After three rinses with phosphate-buffered saline (PBS), the cells were permeabilized in cold 0.2% Triton X-100 for 5 min and incubated with 5% bovine serum albumin/PBS for 30 min. Then, the cells were incubated with an antibody against Spi-C (diluted 1:50) for 2 h at room temperature. After washing three times with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (diluted 1:200; Thermo Fisher Scientific, Rockford, IL, USA) for 1 h in the dark. Then, the nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; diluted 1:1000) for 30 min. After washing with PBS, the cells were observed and imaged using an Olympus CKX41 fluorescence microscope (Olympus, Tokyo, Japan).
10
Evaluation of Tumor Cell Capture Efficiency
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sample preparation and flow test to evaluate cell‐capture efficiency were carried out as described previously.13 In brief, tumor cells were labeled with CellTrace CFSE Cell Proliferation Kit (Life Technologies, Carlsbad, CA, USA), and were suspended either in PBS containing 5% BSA or in the blood sampled from a healthy volunteer at the concentration of 100 and 500 cells/mL. A cell suspension sample of 1 mL, which contains 100 or 500 tumor cells, was applied to the CTC‐chip.
Images and movies of cells in the CTC‐chip were monitored and recorded with a CKX41 fluorescence microscope (Olympus, Tokyo, Japan) and a digital video camera (Sony). The actual number of cells that were sent into the chip (N‐total) was determined by counting the number of cells that passed through the inlet of the CTC‐chip, and the number of captured cells (N‐captured) was also determined by counting CFSE‐labelled cells remained on the CTC‐chip. The cell capture efficiency was represented as N‐captured/N‐total.12 , 13 , 14 Experiments were carried out in triplicate.
Images and movies of cells in the CTC‐chip were monitored and recorded with a CKX41 fluorescence microscope (Olympus, Tokyo, Japan) and a digital video camera (Sony). The actual number of cells that were sent into the chip (N‐total) was determined by counting the number of cells that passed through the inlet of the CTC‐chip, and the number of captured cells (N‐captured) was also determined by counting CFSE‐labelled cells remained on the CTC‐chip. The cell capture efficiency was represented as N‐captured/N‐total.
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!