Lyso-Tracker was used to estimate the number of lysosomes and lysosomal function. Lyso-Tracker Red DND-99 (C1046) was purchased from Beyotime Biotechnology (Shanghai, China). The fluorescence intensity was observed under a ZESIS 880 confocal microscope (Zeiss, Oberkochen, Germany), and representative cells were selected and photographed. For live-cell Lyso-Tracker flow cytometry analysis, cells were grown to about 80% confluence and treated with Lyso-Tracker Red DND-99 for 1 h. Following trypsinization with 0.05% trypsin EDTA (PYG0014, Boster, Biological Technology, Ltd. Wuhan, China) and resuspended in PBS for FACSCalibur Flow Cytometry (BD Biosciences, USA). Flow Cytometry analysis was performed using FlowJo software (OR, USA).
Lysotracker red dnd 99
Manufactured by Beyotime
Sourced in China
LysoTracker Red DND-99 is a fluorescent dye used to label and visualize acidic organelles, particularly lysosomes, within living cells. It is a cell-permeable dye that selectively accumulates in these acidic compartments, allowing researchers to monitor their distribution and dynamics.
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Lab products found in correlation
Glass bottom plate, by Corning (2 mentions)
Tcs sp5, by Leica (2 mentions)
Eclipse ti s, by Nikon (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
A1 hd25, by Nikon (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Phalloidin ifluor 488, by Abcam (1 mentions)
Hypoxia oxidative stress detection kit, by Enzo Life Sciences (1 mentions)
Dna extraction kit, by Beyotime (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Rpmi 1640 medium, by Keygen Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (1 mentions)
Gsh assay kit, by Beyotime (1 mentions)
Endothelial cell growth medium 2 egm 2, by Lonza (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Cd62l, by Thermo Fisher Scientific (1 mentions)
8 protocols using lysotracker red dnd 99
1
Quantifying Lysosomal Dynamics via Microscopy
2
Quantifying Phagosome-Lysosome Fusion in J774A.1 Macrophages
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J774A.1 macrophages were grown on coverslips in 24-well cell culture plates for 12 to 16 h. Then, the cells incubated with 10% heat-inactivated serum were infected with FITC-conjugated BCG at an MOI of 20:1 for 1 h, washed 3 times with DMEM, and incubated with DMEM supplemented with 10% fetal bovine serum (FBS) and 50 nM LysoTracker red DND-99 (product number C1046; Beyotime Biotechnology) for 1 h. After a washing step, the cells were fixed with 4% PFA for 30 min at room temperature. Then, the washed coverslips were mounted on glass microscopy slides with antifade mounting medium (product number P0126; Beyotime Biotechnology). To assess the amount of phagosome-lysosome fusion, approximately 10 to 15 unique images were captured at random under a magnification of ×100 (oil immersion lens) using a Nikon A1HD25 confocal microscope. BCG phagosomes were counted to quantify the percentage of phagosomes that colocalized by means of the LysoTracker staining.
3
Autophagosomes Detection via pEGFP-LC3
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For the detection of autophagosomes, BSR cells were grown to 70% confluence in 35-mm glass-bottomed culture dishes (NEST) and then transfected with 2 μg of pEGFP-LC3 using Lipofectamine 2000 (11668-27, Invitrogen, Carlsbad, CA, USA). The cells were disposed with rapamycin or virus as described above at 12 h post-transfection, and then were fixed with cold absolute ethyl alcohol at 24 h post-treatment. The formation of EGFP-LC3 fluorescence dots was observed using a Leica SP2 confocal system (Leica Microsystems, Wetzlar, Germany). The BTV infection was marked using antibody against the BTV NS1 protein, followed by the corresponding secondary antibody conjugated to TRITC. The cell nucleus was counterstained with DAPI.
To observe the effect of chloroquine (CQ) on LC3 puncta, BSR cells transfected by pEGFP-LC3 were infected with BTV in presence or absence of CQ for 24 h at 12 h post-transfection. The cell nucleus was counterstained with DAPI.
To detect autophagic flux, BSR cells were transfected with pEGFP-LC3, infected with BTV or left untreated as described above. After fixment and permeabilization, the cells were stained with 50 nM LysoTracker Red DND-99 (acidic compartments marker) (C1046, Beyotime) for 30 min. After the cells were washed three times with PBS, they were processed to analyze using a confocal microscope.
To observe the effect of chloroquine (CQ) on LC3 puncta, BSR cells transfected by pEGFP-LC3 were infected with BTV in presence or absence of CQ for 24 h at 12 h post-transfection. The cell nucleus was counterstained with DAPI.
To detect autophagic flux, BSR cells were transfected with pEGFP-LC3, infected with BTV or left untreated as described above. After fixment and permeabilization, the cells were stained with 50 nM LysoTracker Red DND-99 (acidic compartments marker) (C1046, Beyotime) for 30 min. After the cells were washed three times with PBS, they were processed to analyze using a confocal microscope.
4
Breast Cancer and Endothelial Cell Culture
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Mouse Breast Cancer cells (4T1) and Human Umbilical Vein Edothelial cells (HUVEC) were supplied by the Shanghai Institute of Cell Biology (Shanghai, China). Roswell Park Memorial Institute 1640 (RPMI-1640) medium, trypsin-EDTA (0.25%), phosphate buffered saline (PBS, pH = 7.4, 6.8, 5.5) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from KeyGEN BioTECH (Nanjing, China). Endothelial Cell Growth Medium-2 (EGM-2) was supplied from Lonza (Switzerland). Cell Counting Kit-8 (CCK-8), LysoTracker Red DND-99, hydrogen peroxide reagent kit, GSH Assay kit and DNA extraction kit were supplied from Beyotime (Shanghai, China). Hypoxia/Oxidative stress detection kit was purchased from Enzo Life Sciences (New York, America). Fetal bovine serum (FBS) was purchased from Bio-Sciences Limited (Irish). BODIPY581/591-C11, N-acetylcysteine (NAC), GPX4 Recombinant Rabbit Monoclonal Antibody, beta Actin Monoclonal Antibody and Goat anti-Rabbit IgG (H + L) Secondary Antibody (HRP) were purchased from Thermo Fisher Scientific Incorporated (Waltham, America). Phalloidin-iFluor 488 and Anti-gamma H2A.X (Alexa Fluor 647) were obtained from Abcam (United Kingdom). 4T1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS in an incubator at 37 °C with 5% CO2 and HUVEC cells were cultured in EGM-2 under the same culture condition.
5
Synthesis and Characterization of PCL-PEG-PCL Triblock Copolymer for Vaccine Delivery
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PCL-PEG-PCL triblock copolymer was synthesized according to our previous literature36 (link),37 . DOTAP, rhodamine-PE, and MPLA were obtained from Avanti Polar Lipids (Alabaster, AL, USA). OVA and FITC were purchased from Sigma-Aldrich (St. Louis, MO, USA). IMQ was obtained from Tokyo Chemical Industry Company (St. Louis, MO, USA). Recombinant murine TNF-α, IL-4, and GM-CSF were obtained from Peprotech (Rocky Hill, NJ, USA). Fluorochrome-labeled CD11c, CD3, CD8, CD4, CD86, CD80, CD40, CD69, CD44, and CD62L antibodies were purchased from eBioscience (San Diego, CA, USA). Pierce™ BCA protein assay kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). DAPI and Lyso Tracker-Red DND-99 were acquired from Beyotime Biotechnology (Shanghai, China). Female C57BL/6 mice were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were treated according to the protocol approved by Chinese Academy of Medical Science and Peking Union Medical College.
6
Confocal Microscopy of BCG Infection
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Coverslips (Solarbio, China) were placed into specific wells of 24-well plates, and 3 × 105 RAW264.7 cells were cultured in each well in DMEM medium without FBS overnight. Then the RAW264.7 cells were infected with the log phase BCG and stained with FITC at an MOI of 10:1 for 3 h with 10% (v/v) mouse serum in the medium. Serum was collected from the mice 4 weeks after the challenge. The infected cells were washed by blank DMEM thrice and stained with 1:20000 LysoTracker® Red DND-99 (Beyotime, China) for 1 h at 37 °C. After washing, the cells were fixed with 4% PFA for 2 h at room temperature. Coverslips were mounted on glass slides using an antifade mounting medium (Solarbio, China). Images were captured using a Nikon A1 confocal microscope (Nikon, Japan). Ten random fields were selected and counted per condition; at least 250 cells were counted in each field. The percentage of P-L fusions was calculated. The BCG CFUs in every 100 phagolysosomes were counted and calculated for each condition.
7
Cellular Uptake of Multifunctional Nanoparticles
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A total of 2 × 104 of PCSCs were seeded into glass-bottom plates (35 mm, Corning Incorporated) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and were incubated at a final concentration of 50 μg/ml of FITC-labeled GNRs@MSNs@PCM and FITC-labeled AbCD133@GNRs@MSNs@PCM for 24 h. The cells were incubated with 60 nM of LysoTracker Red DND-99 (Beyotime Institute of Biotechnology, Haimen, China) for 1 h at 37°C. After being washed with PBS, the cells were fixed with 4% paraformaldehyde and stained with 10 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI; Sigma). The cells were then washed three times with PBS and mounted. The micrographs were first observed under a Nikon fluorescence microscope (Nikon Eclipse Ti-S, CCD: Ri1) and then under a laser scanning confocal microscope (Leica TCS sp5, Germany) and were used for confocal luminescence imaging with a 63 × oil immersion objective lens.
8
Cellular Uptake of Functionalized Mesoporous Silica Nanoparticles
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A total of 2 × 104 HFLS cells were seeded in glass-bottom plates (35 mm, Corning Incorporated) in DMEM with 10% FBS and incubated at a final concentration of 50 μg/ml FITC-labeled MSNs and FITC-labeled MSNs@CADY for 24 h. The cells were incubated with 60 nM LysoTracker Red DND-99 (Beyotime Institute of Biotechnology, Haimen, China) for 1 h at 37°C. After washing with PBS, the cells were fixed with 4% paraformaldehyde and stained with 10 μg/ml DAPI (4′,6-diamidino-2-phenylindole, Sigma). The cells were washed three times with PBS and mounted. Micrographs were first observed under a Nikon fluorescence microscope (Nikon Eclipse Ti-S, CCD: Ri1) and then under a laser scanning confocal microscope (Leica TCS sp5, Germany) used for confocal luminescence imaging with a 63 × oil immersion objective lens.
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