The colonic primary sensory neurons were labeled in a retrograde manner 10 days before the electrophysiological experiment. The specific method was as follows: rats were anesthetized intraperitoneally with 2% pentobarbital sodium at a dose of 0.25 ml/100 g. The lower abdomen was shaved, and the colon was fully exposed in a sterile environment. A 32G needle connected to a Hamilton microsyringe (10 μl) was used to slowly inject DiI (1 mM, Beyotime Biotechnology Co., Ltd., C1036) into the intestinal wall of the colon. After the injection was done, the needle remained still for 1 min and was then pulled out and wiped with a cotton ball to remove the leaked dye. Injections were conducted at 10 points, with 1 μl per point. After the injection, the rat abdominal cavity was sutured, local disinfection was conducted using iodophor, and the rats were then kept in a single cage for recovery.
C1036
Manufactured by Beyotime
Sourced in China
The C1036 is a laboratory equipment product. It serves as a core function for specific laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
P1585, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
A1r mp, by Nikon (1 mentions)
Tcs sp5 lsm, by Leica (1 mentions)
Ni u fluorescence microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Nis elements software, by Nikon (1 mentions)
C1039, by Beyotime (1 mentions)
C1038, by Beyotime (1 mentions)
Airyscan 2, by Zeiss (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
C1005, by Beyotime (1 mentions)
6 diamino 2 phenylindole dapi, by Beyotime (1 mentions)
7 protocols using c1036
1
Retrograde Labeling of Colonic Sensory Neurons
2
Nano-UCAs Uptake by Macrophages
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To evaluate Nano-UCAs uptake by macrophages, THP-1 cells were seeded in 35-mm 6-well plates at a density of 1×106 cells/well in the presence of 100 nM phorbol myristate acetate (PMA, P1585, Sigma, St Louis, USA) and allowed to attach to the plates for 48 h. After addition of PMA, differentiated THP-1 cells were incubated with 1×1010 particles/mL Nano-UCAs labeled with 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine (DiI) perchlorate (C1036, Beyotime Biotechnology) at room temperature for 1 h. After lysosomal fluorescence (C1046, Lyso-tracker red, Beyotime Biotechnology) and nucleus fluorescence (C1022, Hoechst 33,342 blue, Beyotime Biotechnology) staining, cells were fixed and visualized by the laser confocal fluorescence microscopy (LCFM, A1R-MP, Nikon, Tokyo, Japan). For quantitative analysis, after incubation with DiI-labeled Nano-UCAs, THP-1 cells were detached with trypsin EDTA, collected, and analyzed using a flow cytometer (FACS Caliburl, BD Biosciences, Franklin Lake, USA). The labeled Nano-UCAs were also incubated with non-phagocytic NCI-H1299 cells and observed using LCFM, as described above.
3
Zebrafish Tumor Migration and Angiogenesis Assays
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We used Tg (fli1a: EGFP) transgenic zebrafish for the tumour migration assay. Approximately 300 cells that had been stained with DiI (C1036, Beyotime, China) were injected into the perivitelline cavity of 48 h postfertilization zebrafish embryos with a microinjection system. DiI-stained cells were visualized under a fluorescence microscope.
For angiogenesis assays in zebrafish, the FGF19 overexpression plasmid was injected into 1–2-cell stage fertilized eggs of Tg (fli1a: EGFP) transgenic zebrafish. Seventy-two hours later, we observed the morphology of subintestinal vessels (SIVs) with a confocal microscopy (TCS-SP5 LSM, Leica, Germany).
For angiogenesis assays in zebrafish, the FGF19 overexpression plasmid was injected into 1–2-cell stage fertilized eggs of Tg (fli1a: EGFP) transgenic zebrafish. Seventy-two hours later, we observed the morphology of subintestinal vessels (SIVs) with a confocal microscopy (TCS-SP5 LSM, Leica, Germany).
4
Fluorescent Labeling of Exosomes
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Exosomes were labeled with a red fluorescent lipophilic dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Beyotime, C1036), allowing monitoring of exosome movement. DiI emits strong fluorescence when excited by green light and incorporated into membranes, and does not disrupt the membrane properties. Exosomes were resuspended in sterile PBS and incubated with 5 μM DiI for 10 min. DiI-exosomes were then washed and resuspended in sterile PBS three times to remove free DiI and other impurities such as lipoproteins. The control was prepared by DiI incubation with PBS, which was washed as for the DiI-exosome preparation.
5
Quantitative Infection Assay for APP and STM
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For APP or STM coinfection, cells were washed with PBS to remove unattached bacteria and then lysed in 0.1%Triton X-100 for 5 min, the lysate was serially diluted in PBS and plated onto TSB or LB-Agar for overnight before bacterial colonies was counted and colony formation unit (CFU) in each plate was calculated. GFP positive cells were analyzed by flow cytometry after washing with PBS. For immunofluorescence assay, cells were fixed with 4% paraformaldehyde for 20 min at room temperature after removal of unattached bacteria and were stained with a red fluorescence probe Dil (C1036, Beyotime, China) and DAPI (C1005, Beyotime, China) to label cell membrane and nucleus, respectively. Images were acquired on a Nikon Ni-U fluorescence microscope using Nis-elements software (Nikon, Japan).
6
FRET-based Confirmation of PMV-M2EV Fusion
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To confirm the fusion of PMV and M2EV, we employed Förster resonance energy transfer (FRET) [26 ]. Platelet membrane vesicles (PMV) were labeled with 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine,4-Chlorobenzenesulfonate Salt (DiD, excitation/emission = 644/665 nm, C1039, Beyotime) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, excitation/emission = 549/565 nm, C1036, Beyotime). These labeled PMV were extruded with M2EV at various protein weight ratios (PMV:M2EV) of 1:0, 1:1, 1:2, and 1:3. Then, the fluorescence spectra were collected within the 550–750 nm range with excitation at 500 nm using a Spark Multimode Microplate Reader (Tecan, Switzerland). To assess membrane co-localization, M2EV was exposed to 2 μg/mL DiD at a temperature of 37 °C for 30 min. Then, it was subjected to centrifugation at a speed of 100,000×g for 70 min in order to eliminate excess dye. PMV was treated with 2 μg/mL 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO, excitation/emission = 484/501 nm, C1038, Beyotime) for a duration of 30 min. After that, it was subjected to centrifugation at a speed of 8000×g for a period of 15 min. Fused P-M2EV and the mixtures of PMV and M2EV were observed using the confocal fluorescence microscopy (LSM980 with Airyscan2, Carl Zeiss AG, Germany) with a 100× oil objective.
7
Exosome Uptake by H9c2 and HUVEC
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To demonstrate the uptake of Exos by H9c2 cells and HUVEC, Exos were labeled with DiI (red fluorescent dye, C1036, Beyotime, China) and co-cultured with recipient cells at 37 °C for 6 or 24 h, washed with PBS, and fixed with 4% paraformaldehyde for 20 min. The nuclei were stained with 6-diamino-2-phenylindole (DAPI) (0.5 g/ml, C1005, Beyotime, China) for 10 min, and observed using a confocal microscope.
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