RAW264.7 cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin at 5% CO2 and 37°C. The exosomes were labelled with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, MO, USA) according to the manufacturer's protocol, and washed 5 times with PBS. The last PBS supernatant collected after exosome labelling was used as control. The cells were incubated with exosomes (10 μg/ml) for 12 h. After being washed with PBS, the cytoskeleton was stained with phalloidin (YEASEN Biotech, Shanghai, China) and the nucleus with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA). Fluorescence was analysed by a LEICA TCS-SP2 laser confocal microscope (Leica Microsystems, Wetzlar, Germany).
Tcs sp2 laser confocal microscope
Manufactured by Leica
Sourced in Germany
The TCS SP2 laser confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a modular design, allowing for the integration of various laser sources and detection systems. The TCS SP2 is capable of performing optical sectioning, multi-channel fluorescence imaging, and time-lapse studies with high resolution and sensitivity.
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Lab products found in correlation
Pkh26 red fluorescent cell linker kit, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Pam 2500, by Walz (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem and rpmi 1640 cell culture media, by Thermo Fisher Scientific (1 mentions)
Jem 100sx transmission electron microscope, by JEOL (1 mentions)
Gapdh, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Caspase 9, by Abcam (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Abcam (1 mentions)
Disuccinimidyl suberate, by Sangon (1 mentions)
Anti mouse igg alexa594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
12 protocols using tcs sp2 laser confocal microscope
1
Exosome Uptake Visualization in RAW264.7 Cells
2
Quantitative Immunolocalization of Histone Modifications
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Interphase nuclei prepared from squashed root tips, as described in Forestan et al. (2013) (link), were immunostained with specific antibodies against histone modifications and Alexa Fluor 488-conjugated secondary antibodies (Life Technologies). Immunolocalization experiments were carried out in triplicate and for each genotype × antibody combination at least eight squashed root tips were analyzed. Nuclei were counterstained with propidium iodide in Vectashield Mounting Medium (Vector) and observed with a Leica TCS SP2 laser confocal microscope (Leica Microsystems, Heidelberg, Germany). Microscope images were then analyzed and quantified using CellProfiler automated image analysis software (Carpenter et al. 2006 (link)). Nuclei intensity for both antibody and propidium iodide signal was calculated from at least 200 nuclei (on different slides of the three replicates) for each antibody × genotype combination. Further detailed methods for nuclei isolation, antibody dilutions, and Western blot analysis are fully provided in Supplemental Materials and Methods in File S16 .
3
Chlorophyll Content Determination in Fruit and Leaves
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Chlorophyll content was determined in the fruit pericarp and leaves according to the methods described by Powell et al. (2012) (link). The experiments were performed using 2-month-old plants and the leaf samples were selected from the fourth internode counting from the shoot tip. For the determination of chlorophyll autofluorescence, the pericarp was peeled off the fruit and observed under a TCS SP2 laser confocal microscope (Leica, Germany).
4
Visualizing TRIM30α and STING in L929 Cells
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L929 cells were transfected with expressing plasmids for cyan fluorescent protein-labeled TRIM30α and yellow fluorescent protein-labeled STING. After 24 h, cells were stimulated for 4 h with 1 μg/ml poly(dA:dT), 1 μg/ml ISD or left unstimulated. After stimulation, cells were fixed with 4% PFA in PBS and permeabilized with Triton X-100 and then blocked with 10% FBS in PBS, stained with anti-calnexin, followed by Alexa Fluor 647 donkey anti-rabbit IgG. Nuclei were stained with 4, 6-diamidino-2-phenylindole, and fluorescent images were captured with a Leica TCS SP2 laser confocal microscope.
5
Chlorophyll Quantification in Tomato
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For chlorophyll content determination, the fruits at different developmental stages and leaf tissues were collected and examined based on the methods described by Powell et al. (2012)39 (link). To determine chlorophyll autofluorescence, pericarp was peeled off tomato fruits and observed with a TCS SP2 laser confocal microscope (Leica, Germany). For transmission electron microscopy, pericarp tissues were examined with an FEI Tecnai T12 twin transmission electron microscope according to the method described by Nguyen et al. (2014)8 (link).
For measurements of photosynthesis rates, the green mature fruits and leaves were measured via a PAM-2500 pulse-amplitude modulation fluorometer (Heinz Walz, Effeltrich, Germany). The chlorophyll fluorescence parameter was measured based on the method described by Maury et al. (1996)61 (link).
For measurements of photosynthesis rates, the green mature fruits and leaves were measured via a PAM-2500 pulse-amplitude modulation fluorometer (Heinz Walz, Effeltrich, Germany). The chlorophyll fluorescence parameter was measured based on the method described by Maury et al. (1996)61 (link).
6
Rapamycin-Induced MDC Staining Protocol
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For MDC staining, tissues were induced with exogenous rapamycin, fixed in 4% paraformaldehyde, and washed in PBS buffer by three times. Tissues were stained with 0.2 mM monodansylcadaverine (MDC) for 30 min and sectioned with a freezing microtome. Sections were analysed under a Leica TCS SP2 laser confocal microscope (excitement at 405 nm and detection at 445 to 465 nm).
7
Esophageal Cancer Cell Apoptosis Assay
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Eca-109 human esophageal cancer cells and HPT-1A normal human esophageal epithelial cells were provided by the Cell Resource Center of the Shanghai Life Sciences Institute, Chinese Academy of Sciences (Shanghai, China). Other materials included CPI-455 (MSDS, 1628208-23-0; Gibco, Ltd., United States), DMEM and RPMI 1640 cell culture media and fetal bovine serum (Gibco, Ltd). Anti-human P53 (cat no. sc-6243), Bax, KDM5C (cat no. sc-81623 ), Caspase-9 (cat no. 9746), Caspase-3 (cat no. 9502), GAPDH (glyceraldehyde-3-phosphate dehydrogenase, cat no. sc-47778) polyclonal antibody (1:200; Abcam, United Kingdom) were used for western blotting. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits (cat no. F7250) were from Sigma-Aldrich (United States). polyvinylidene difluoride (PVDF) membranes were from Biyuntian Biotech (China) and bicinchoninic acid (BCA) protein quantitative detection kits were from Thermo Fisher (United States). An Epics Ultra flow cytometer (Beckman Coulter, United States), JEM-100sx transmission electron microscope (Jeol Ltd., Japan), and a TCS SP2 laser confocal microscope (Leica, Germany) were used.
8
Immunoblot Analysis of ASC Oligomerization in BMDMs
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For immunoblot of ASC oligomerization, BMDMs were lysed with Triton Buffer (pH 7.5 50 mM Tris HCl, 150 mM NaCl, 0.5% Triton X‐100, and 0.1 mM PMSF) for 10 min on ice. The cell lysates were centrifuged at 6,000×g for 15 min at 4°C and then resuspended in Triton Buffer and disuccinimidyl suberate (2 mM) (Sangon Biotech). After incubation for 30 min at 37°C for cross‐linking, cell pellets were washed by Triton Buffer for two times. Then cell lysates were centrifuged at 12,000×g for 15 min at 4°C, and were redissolved in 1× SDS loading buffer (Sangon Biotech) for immunoblot assays. For IF analysis of ASC speck formation, BMDMs washed with cold PBS three times, fixed in paraformaldehyde (4%, v/v) for 10 min, permeabilized with Triton X‐100 (0.1%, v/v) for 15 min, and blocked with BSA (3%, v/v). Cells were then stained with ASC antibody (1:200) overnight at 4°C and stained with Alexa fluor 488‐labeled secondary antibody (1:200) (Abcam) for 1 h at room temperature. Last, DAPI was used to stain cell nuclei. Cells were visualized using TCS SP2 confocal laser microscope (Leica).
9
Immunofluorescence Staining of KAT8
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Macrophages were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 5% BSA, cells were labeled with anti-KAT8 antibody followed by staining with corresponding secondary antibodies. Cells were observed with a Leica TCS SP2 confocal laser microscope.
10
Hypoxia and LPS effects on YTHDF3
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HTR8/SVneo cells plated on glass coverslips in six-well plates were treated in hypoxia condition for 12 h or stimulated with LPS for 6 h as indicated and fixed in 4% paraformaldehyde. Cells were then stained with YTHDF3 antibody and viewed using a Leica TCS SP2 confocal laser microscope.
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