Yeast cells were cultured in SC medium (0.17% YNB, 2% glucose, and 0.2% amino acids) at 30°C. When gowning to log phase, yeast cells were collected and then washed twice in stilled water, cultured in SD‐N media for indicated times at last. Aliquots of liquid culture were collected at indicated time points and allowed to precipitate on concanavalin A coated cover glass for 5 min. Images were captured by Zeiss Observer 7 machine equipped with Apotome. Representative images from at least three independent repeated experiments were shown. HEK293T cells were grown in confocal wells and observed by Zeiss Observer 7 machine equipped with Apotome. Representative images from at least three independent repeated experiments were shown.
Observer 7
Manufactured by Zeiss
Sourced in Germany
The Observer 7 is a high-performance light microscope designed for a wide range of applications in the life sciences and materials research. It features advanced optics, flexible configuration options, and user-friendly controls to support precise observation and analysis.
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Lab products found in correlation
Airyscan, by Zeiss (2 mentions)
96 well tissue culture plate, by BD (1 mentions)
Cfda se, by Beyotime (1 mentions)
Live dead viability cytotoxicity kit for mammalian cells, by Thermo Fisher Scientific (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Alp stain working solution, by Beyotime (1 mentions)
Dapi fluoromount, by Southern Biotech (1 mentions)
Ab1542, by Merck Group (1 mentions)
Mouse anti cd68, by Abcam (1 mentions)
Rabbit monoclonal anti zo 1, by Abcam (1 mentions)
Lsm 800 confocal, by Zeiss (1 mentions)
Formaldehyde suspension, by Carl Roth (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Orca falsh4 camera, by Hamamatsu Photonics (1 mentions)
Zen blue v2, by Zeiss (1 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Zen pro software, by Zeiss (1 mentions)
18 protocols using observer 7
1
Yeast and HEK293T Cell Imaging
2
Evaluating Cell Viability with DFO Hydrogels
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Alamar Blue (AB) and Live/Dead assays were conducted to evaluate the effect of DFO@G and DFO@G-QCSFP hydrogels on cell viability and proliferation. Briefly, human umbilical vein endothelial cells (HUVECs) were seeded in a 48-well plate at a density of 1 × 104 cells per well. After the HUVECs adhered to the plate for 24 h, 500 μL of the sample was added into each well to incubate for different time intervals. Finally, cell viabilities were quantified using AB assay and normalized to the control group. Then, Live/Dead staining was performed by using a 12-well plate with a density of 2 × 104 cells per well. After cells adhered to the plate for 24 h, 500 μL hydrogel was added and incubated for different time intervals. After staining with 500 μL of calcein-AM/propidium iodide dye for 15 min, cells were observed under a fluorescent microscope (Carl Zeiss Observer 7, Germany) for the green and red fluorescence.
3
Cell Viability and Proliferation Assays
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The cell viability was evaluated using a LIVE/DEAD assay kit according to the manufacturer's instructions. Calcein AM (0.5 μL/mL) and ethidium homodimer-1 (2 μL/mL) were diluted in DPBS to form the staining solution, which was added to the well after removing the culture medium. Then, the cells were incubated for 30 min at 37 °C in the dark. Live (green stain) and dead (red stain) cells were imaged using an inverted fluorescence microscope (Observer 7, Zeiss, Germany) on days 1, 3 and 5 of culture.
L929 cells were seeded in 96-well tissue culture plates (BD Biosciences) at 5000 cells/cm2. The leaching medium was prepared by immersing G20OD5 in complete medium at 37 °C for 48 h. The proliferation of L929 cells was quantitatively determined by the CCK-8 assays. The absorbance was measured at 450 nm with a microplate reader at 1, 3 and 5 days of culture.
L929 cells were seeded in 96-well tissue culture plates (BD Biosciences) at 5000 cells/cm2. The leaching medium was prepared by immersing G20OD5 in complete medium at 37 °C for 48 h. The proliferation of L929 cells was quantitatively determined by the CCK-8 assays. The absorbance was measured at 450 nm with a microplate reader at 1, 3 and 5 days of culture.
4
Tracking Stem Cell Distribution
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hUC-MSCs (1 × 108) were labelled with 5(6)-Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (C0051, Beyotime, Shanghai, China). The labeled cells were washed three times with PBS and injected into 24 mice via tail vein 24 h after PTX treatment. The distribution of hUC-MSCs in the mice heart, liver, spleen, lung, kidney, and testis was detected at 8 different time points within 72 h (15 min, 30 min, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h). Fluorescence signals in frozen sections (5 μm) were detected by a fluorescence microscope (Observer 7, Carl Zeiss, Jena, Germany) at a magnification of × 20.
5
Apoptosis assessment via TUNEL assay
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A commercial terminal TUNEL (Roche, Germany) was purchased to examine the apoptosis. The assay was performed according to the manufacturer’s instructions. Images were captured using fluorescence microscopy (Observer 7, Zeiss).
6
Evaluating Cell Viability by LIVE/DEAD Assay
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The viability of cells was evaluated using a LIVE/DEAD® Viability/Cytotoxicity kit for mammalian cells (Invitrogen™) according to the manufacturer’s instructions. Briefly, cells were incubated with 0.5 μL/mL calcein AM and 2 μL/mL ethidium homodimer-1 (EthD-1) in DPBS for 15 min at 37 °C in the cell culture incubator to allow simultaneous staining of living and dead cells. At 1, 3, and 5 days post-seeding, live (green-stained) and dead (red-stained) cells were imaged using an inverted fluorescence microscope (Observer 7, Zeiss, Germany). The numbers of live and dead cells were quantified using ImageJ software. Viability was then calculated using the following equation: Cell viability (%) = Living cells/ (Living cells + Dead cells) × 100%.
7
Visualization of Toxoplasma gondii PVM in Ileum
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Antigen retrieval in deparaffinized paraformaldehyde-fixed ileum tissue sections from wt and Ifnlr1−/− mice was performed with 0.01 M sodium citrate buffer as previously described13 . Slides were blocked with 10 % normal donkey serum (Jackson ImmunoResearch) and stained o/n with rat anti GRA7 (T. gondii PVM marker) and E-Cadherin (Cell Signalling) followed by 1 h incubation with the appropriate Cy3-, or Cy5-conjugated secondary antibodies and DAPI. Slides were mounted in Fluor Save Reagent (Calbiochem). Tissue sections were visualized using a Zeiss Axioplan 2 non-inverted fluorescence microscope.
ODMs were infected with T. gondii ME49 for 2 h at MOI 4. Monolayers were washed two times with PBS and fixed for 30 min at RT with 4 % PFA. Cells were permeabilized and stained as previously described14 . Antibodies and dilutions are listed inTable 1 . Intensities were determined by taking the average of 4 intensity values along 2 lines crossing the PV perpendicularly subtracted by the respective background fluorescence, as described previously80. The measurements were done using the Fiji/ImageJ software with a custom macro (code can be found at https://github.com/Kartoffelecke/PVM-profiler ). Pictures were taken on the Zeiss Observer 7 with a 40x magnification.
ODMs were infected with T. gondii ME49 for 2 h at MOI 4. Monolayers were washed two times with PBS and fixed for 30 min at RT with 4 % PFA. Cells were permeabilized and stained as previously described14 . Antibodies and dilutions are listed in
8
Osteogenic Differentiation of GH-NbBG in mBMSCs
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Mouse bone marrow mesenchymal stem cells (mBMSCs) were used to investigate the osteogenic differentiation of GH-NbBG. Similarly, the mBMSCs were cultured with Control, BG and NbBG extracts. The medium was replaced every 2–3 days. Alkaline phosphatase (ALP) activity was employed to investigate the osteogenic differentiation potential at day 7. The mBMSCs were fixed with 2.5% glutaraldehyde for 15 min and washed with PBS, and incubated in ALP stain working solution (Beyotime, China) for 30 min at room temperature. The reaction was stopped by removing the ALP stain working solution and rinsing with PBS. Stained cells were visualized with light microscope (Observer7, Zeiss, Germany).
The osteogenic differentiation mechanism analysis of GH-NbBG were researched by bioinformatic analysis and RNA sequencing (RNA-seq). In order to verify the correctness of mechanism speculation, the protein expressions of RUNX2 and OCN were analyzed by western bloting and immunofluorescent staining. The detailed experimental procedures were provided in supplementary data.
The osteogenic differentiation mechanism analysis of GH-NbBG were researched by bioinformatic analysis and RNA sequencing (RNA-seq). In order to verify the correctness of mechanism speculation, the protein expressions of RUNX2 and OCN were analyzed by western bloting and immunofluorescent staining. The detailed experimental procedures were provided in supplementary data.
9
Quantitative Immunofluorescence of Tubb3 and TH
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Immunofluorescence for Tubb3 (52623; Abcam) and TH (AB1542; EMD Millipore) was performed as previously described (17 (link)). Dilutions used were 1:200 for TH and 1:800 for Tubb3, with 1:500 used for secondary antibodies. Sections were mounted with DAPI Fluoromount (0100-20; SouthernBiotech) and imaged using a Zeiss Observer 7 at 40× magnification. Fluorescent signal in each image layer was quantified using Fiji automatic signal measurement prior to any adjustment of image brightness or contrast and normalized to the average of the control signal (64 (link)). The final resolution of the 40× images was 1,920 × 1,210 with a pixel width of 0.1465 microns.
10
Immunocytochemical Characterization of Cells
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Immunostaining was carried out to confirm cell-specific and tissue-specific markers. For this purpose, the cultured cells or cryostat sections were washed with PBS and fixed with 4% paraformaldehyde for 15 min and then washed twice with PBS. Then, they were incubated in PBS-3% bovine serum albumin (BSA)-0.3% Triton X-100 at room temperature for 1 h followed by incubation with primary antibodies (rabbit polyclonal anti-OCT4, 1:400, CST; mouse polyclonal anti-Sox2, 1:200, Abcam; rabbit monoclonal anti-ZO-1, 1:400, Abcam; mouse polyclonal anti-Mitf, 1:400, Santa Cruz; rabbit anti-human nuclear antigen, Abcam; mouse anti-CD68, Abcam; rabbit polyclonal anti-Brn3b, 1:200, Proteintech; rabbit polyclonal anti-RPE65, Abcam) overnight at 4 °C. Following this, the cells or cryostat sections were incubated with goat anti-mouse IgG H&L Alexa Fluor®488/goat anti-rabbit IgG H&L Alexa Fluor®555 (1:500, Abcam) for 2 h at room temperature. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Images were captured with fluorescence microscopy (Observer 7, Zeiss).
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