HeLa cells and COS7 cells were obtained from ATCC. Mouse fibroblast 17Cl-1 cells were kindly gifted by Dr. Hongyu Deng’s lab (IBP, CAS). Vero E6 cells were kindly gifted by Dr. Yan Li (Institute of Microbiology, CAS). The HeLa cell line with stable TFEB-GFP expression was kindly gifted by Dr. Richard Youle’s lab (NINDS). All of the cell lines were maintained in DMEM (SH30022.01B, Hyclone) with 10% FBS (SH30084.03, Hyclone) and 50 mg/ml penicillin-streptomycin at 37 °C and 5% CO2. For amino acid and HBSS starvation, cells were incubated with DMEM without amino acids (SH4007.01, Hyclone) or HBSS (14025-092, Gibco) for the indicated time. For Brefeldin A (BFA) treatment, cells were incubated with 5 μg/mL BFA (S1536, Beyotime) at 37 °C for 6 h. For chloroquine diphosphate salt (CQ) treatment, cells were incubated with 100 mg/ml CQ (C6628, Sigma) at 37 °C for 4 h. For Torin 1 (14379, Cell Signaling Technology) treatment, cells were incubated with 1 μM Torin 1 at 37 °C for 4 h. For LysoTracker staining, samples were incubated with LysoTracker (L7528, Life Technologies) at 37 °C for 30 min according to the manufacturer’s instructions.
Lysotracker
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, Germany
LysoTracker is a fluorescent dye that selectively labels acidic organelles, such as lysosomes, in live cells. It is a useful tool for the visualization and analysis of lysosomal function in various cell types and biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker, by Thermo Fisher Scientific (36 mentions)
Hoechst 33342, by Thermo Fisher Scientific (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (12 mentions)
Er tracker, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Hoechst, by Thermo Fisher Scientific (7 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (6 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Lipidtox, by Thermo Fisher Scientific (4 mentions)
Mitotracker red, by Thermo Fisher Scientific (4 mentions)
Zen software, by Zeiss (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Bafilomycin a1, by Merck Group (4 mentions)
Lysotracker, by Zeiss (4 mentions)
Mitosox, by Thermo Fisher Scientific (4 mentions)
Cellrox, by Thermo Fisher Scientific (4 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (4 mentions)
Dm6b fluorescence microscope, by Leica (4 mentions)
Dq ova, by Thermo Fisher Scientific (3 mentions)
310 protocols using lysotracker
1
Cell Culture Protocols for Various Cell Lines
2
Isolation of Heterotrophic Nanoflagellates
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The HNF described here was isolated in a previous culturing effort (del Campo et al. 2013) , and the isolation procedure is explained below. Seawater from the Blanes Bay Microbial Observatory sampled on September 30 th , 2008 was filtered through 3 µm and sent to Bigelow Laboratory for Ocean Sciences (Boothbay Harbor, ME, USA) for cell sorting in a MoFlo™ Flow Cytometer (Dako-Cytomation, Denmark). Digestive vacuoles of heterotrophic protists were stained using the vital stain LysoTracker® (Life Technologies, NY, USA) and cells were sorted based on their green fluorescence (LysoTracker® fluorescence) and the absence of chlorophyll fluorescence.
Details of the staining protocol and flow cytometer setup are described elsewhere (Rose et al. 2004; Heywood et al. 2011) . Side scatter was used to select only the smallest protists, approximately <10 µm in diameter. Individual target cells were deposited into 24-well plates, in which some wells were dedicated for positive controls (10 cells per well) and negative controls (0 cells per well). All wells on the microplates contained 1ml aged seawater together with natural bacteria at a final concentration of 5 X 10 6 bacteria ml -1 . Multi-well plates were hand carried back to the Institut de Ciències del Mar (Barcelona, Catalonia, Spain) by plane at room temperature (12 hours).
Details of the staining protocol and flow cytometer setup are described elsewhere (Rose et al. 2004; Heywood et al. 2011) . Side scatter was used to select only the smallest protists, approximately <10 µm in diameter. Individual target cells were deposited into 24-well plates, in which some wells were dedicated for positive controls (10 cells per well) and negative controls (0 cells per well). All wells on the microplates contained 1ml aged seawater together with natural bacteria at a final concentration of 5 X 10 6 bacteria ml -1 . Multi-well plates were hand carried back to the Institut de Ciències del Mar (Barcelona, Catalonia, Spain) by plane at room temperature (12 hours).
3
Immunostaining of HeLa Cells
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HeLa cells cultured on coverslips in 24-well cell culture plates were transfected with the indicated plasmids and cultured for 24 h before the next operation. After 3 washes with PBS buffer, the cells were fixed with 4% polyformaldehyde for 20 min and permeabilized with 100 mg/ml digitonin (D141, Sigma) for 10 min at room temperature with gentle shaking. For immunostaining with anti-TFEB and anti-MED1 antibodies, cells were permeabilized with 0.3% Triton X-100 (9002-93-1, Amresco) for 20 min at room temperature with gentle shaking. After blocking with 5% goat serum for 60 min, cells were incubated with the indicated primary antibodies (diluted in 5% goat serum) for 1.5 h at room temperature. The cells were then washed with PBS 3 times and stained with fluorescentlylabeled secondary antibodies for 1 h at room temperature. For LysoTracker staining, the cells were incubated with LysoTracker (L7528, Life Technologies) in a 37 C cell culture incubator for 30 min according to the manufacturer's instructions before fixation.
After 3 washes with PBS, the coverslips were mounted with DAPI in 50% glycerol. The images were acquired using a confocal microscope.
After 3 washes with PBS, the coverslips were mounted with DAPI in 50% glycerol. The images were acquired using a confocal microscope.
4
Quantifying Small Eukaryote Abundances
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In this study, “small heterotrophic eukaryotes” included pico and nano eukaryotes, and refers mainly to small HNF. Small eukaryote abundances were estimated following the protocol by Rose et al. (2004 (link)). From a stock solution of 1 mM Lysotracker Green (Molecular Probes), 1 μl was added to 99 μl of <0.2 μm MilliQ, and 3.8 μl of this diluted Lysotracker were added to 0.5 ml of the sample, ending at 75 nM Lysotracker final concentration. We analyzed the samples as in Rose et al. (2004 (link)), using a combination of light scatter (SSC) and green (FL1) and red (FL3) fluorescence. Samples were run alive, less than 3 h after sample collection, and at high (ca. 100 μl min−1) speed. Concentrations were obtained from weight measurement of the volume analyzed.
5
Antigen Uptake and MHC-I Trafficking
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To examine antigen uptake and processing, 1 μg/mL DQ-OVA (Molecular Probes), which is OVA conjugated to a self-quenched dye that emits green fluorescence upon degradation, was mixed with 400 μg/mL of an adjuvant and incubated for 16 h at 4 °C. DCs were plated onto μ-Slides (Ibidi GmbH) and stimulated with the mixture for 4 h at 37 °C followed by incubating with LysoTracker and DAPI (Molecular Probes). To examine the accumulation of MHC-I in acidic compartments, DCs were incubated with a mixture of OVA-Texas red (1 μg/mL; Molecular Probes) with either alum or PC nanogel for 3 h at 37 °C and then exposed to LysoTracker for the last 30 min. The cells were fixed, permeabilized and stained with an anti-MHC-I (BD) followed by Alexa488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were counterstained with DAPI. The stained cells were examined with an LSM5 Pascal confocal fluorescence microscope (Carl Zeiss). The percentages of cells in which MHC-I colocalized with LysoTracker or OVA (Manders’ coefficient) in the insets were calculated using the JACoP plugin of the ImageJ software.
6
Lysosome Dynamics in Francisella Infection
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For LysoTracker staining and colocalization with F. novicida, WT and Ctsb−/− BMDMs were infected with GFP-expressing F. novicida and followed by labeling with 100 nM LysoTracker (L-7528; Molecular Probes) for the last 30 min. Cells were washed and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were washed three times and mounted using mounting medium (H-1200; Vector Laboratories). For LC3B, LAMP1, and TFEB immunostaining, uninfected and infected BMDMs were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were then permeabilized with cold methanol for 5 min, washed with PBS, and blocked in 1× ELISA buffer with 0.1% saponin for 1 h. Cells were stained with anti-LC3B (NB600-1384; Novus Biologicals), anti-LAMP1 (14-1071-85; eBioscience), or anti-TFEB (A303-673A; Bethyl Laboratories, Inc.), all at 1:500 dilution, overnight at 4°C. Cells were washed, stained with fluorescence-conjugated secondary antibody for 1 h, and mounted using mounting medium (H-1200; Vector Laboratories). Cells were observed on a confocal microscope (Axio Observer; Z1; SlideBook 6 software; ZEISS) for image acquisition and data analysis.
7
Immunofluorescence Imaging of HER2, LAMP1, and MHC-II
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293T, E0771, and JAWSII cells were grown on 8-well slides (MatTek) coated with 1 µg/mL human fibronectin (EMD Millipore). Transfection was performed using Lipofectamine3000 (Thermofisher). For Lysotracker (ThermoFisher) staining, cells were incubated with 75 nm Lysotracker for 1 hour at 37°C. For endoplasmic reticulum (ER) staining, cells were incubated with 2 μL ER CellLight ER-RFP Bacman V.2.0 (Thermofisher) for 24 hours. Slides were washed with phosphate buffered saline (PBS), fixed with 10% formalin for 20 min and mounted with DAPI Fluoromount-G (SouthernBiotech). Staining was performed in 0.1% PBST with rabbit anti-HER2 (1:200, Cell Signaling), rat anti-LAMP1 (1:200, Abcam) or rat anti-MHCII (1:200, Thermofisher) at 4° overnight and antirabbit Alexa Fluor 488 (1:1000 dilution, Invitrogen) or antirat Texas Red-X (1:1000, invitrogen) at room temperature for 1 hour. Slides were mounted with Fluoromount-G with DAPI (Southern Biotech).
8
Lyso Tracker Apoptosis Detection in Embryos
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E11.5 WT and CKO embryos were stripped from the uterus and extra-embryonic membranes were removed. The embryos were then incubated in Lyso tracker (Thermo Fisher Scientific) staining solution (150 nM Lyso tracker in Hanks balanced salt solution) at 37 °C for 45 min. The embryos were fixed in 4% paraformaldehyde overnight and exposed under a stereoscopic microscope with fluorescence (Zeiss Axio Zoom v16) to detect apoptotic signals.
9
Quantifying Lysosomal Proteolysis in T Cells
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For studying lysosomal proteolysis, activated naïve CD4+ T cells were treated with DQ-BSA (5 μg/ml; D12050, Thermo Fisher Scientific) diluted in prewarmed medium and incubated at 37°C for 5 hours. Cells were then briefly washed once with ice-cold PBS containing 2% FBS and kept on ice. Fluorescence of cleaved BSA-DQ was analyzed by flow cytometry. For quantitatively analyzing LysoTracker staining, naïve CD4+ T cells were treated with 100 nM LysoTracker (L7528, Thermo Fisher Scientific) diluted in prewarmed medium, incubated for 2 min at 37°C, and then washed with ice-cold PBS containing 2% FBS and kept on ice. LysoTracker fluorescence was analyzed and quantified by flow cytometry.
10
Multiparameter Analysis of Murine and Human Cells
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Murine cells were stained with LIVE/DEAD (Invitrogen), blocked with mouse serum and anti-murine CD16/32 (clone 2.4G2, Biolegend), and stained for cell surface markers (see Table S1 for a list of antibodies used). Where lysotracker was used, cells were incubated in RPMI with lysotracker (ThermoFisher, 1/2000) for 30 min at 37 °C and washed in fluorescence-activated cell sorting (FACS) buffer prior to staining for surface markers. For LipidTox staining, cells were first fixed in Neutral buffered formalin (NBF; 10%, Sigma), then stained with LipidTox (ThermoFisher, 1/200) for 30 min at room temperature. Human samples were blocked with serum, stained for cell surface markers (see Table S1 for a list of antibodies used), and DAPI was added to the cells prior to acquisition. All samples were acquired using a FACSDiva software 6.3.1, BD Biosciences software, and analyzed with the FlowJo 10 software (Tree Star). For BODIPY LDL uptake experiments, AT cells were preincubated with anti-CD36 IgA (clone CRF D-2712, BD Pharmingen, 22.5 μg/ml) or anti-Tim4 IgG2a (clone RMT4-53, BioXCell, 22.5 μg/ml) or IgG2a control (clone BE0089, BioXCell, 22.5 μg/ml) prior to incubation with BODIPY FM LDL (10 μg/ml, Invitrogen) for 1 h.
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