Lysotracker

Manufactured by Enzo Life Sciences

Sourced in Germany

LysoTracker is a fluorescent probe used to label and track lysosomes in live cells. It selectively accumulates in acidic organelles such as lysosomes due to their low pH environment.

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Lab products found in correlation

4 protocols using lysotracker

Comprehensive Antibody Collection for Cell Signaling

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Antibodies used in this study included anti-STING (13647; Cell Signaling Technology), anti-Flag M2 (F3165; Sigma-Aldrich), anti-HA (H3663; Sigma-Aldrich), anti-EGFP (GL-8; Clontech), anti-LC3 (7543; Sigma-Aldrich), anti-p62 (PM045; MBL), anti-STX17 (HPA001204; Sigma-Aldrich), anti-SNAP29 antibody (111303, SYSY, or sc-135564; Santa Cruz Biotechnology), anti-LAMP1(L1418; Sigma-Aldrich), anti-LAMP2 (sc-18822; Santa Cruz or L0668; Sigma-Aldrich), anti-Myc (9E10, DSHB), anti-WIPI2 (ab105459; Abcam), anti-FIP200 (17250; ProteinTech), anti-ATG16L (PM040; MBL), anti-α-Tubulin (E7; DSHB), anti-VAMP8 (ab76021; Abcam), anti-GABARAP (ab109364; Abcam), anti-AMPK (2532; Cell Signaling Technology), anti-p-AMPK T172 (2535; Cell Signaling Technology), anti-TBC1D1(66433; Cell Signaling Technology), anti-p-TBC1D1 Ser237(07-2268; Sigma-Aldrich), anti-Raptor (2280; Cell Signaling Technology), anti-p-Raptor Ser792 (2083; Cell Signaling Technology), anti-TSC2 (4308; Cell Signaling Technology), anti-p-TSC2 Ser1387 (5584; Cell Signaling Technology), anti-ACC (3662; Cell Signaling Technology), anti-p-ACC Ser79 (3661; Cell Signaling Technology), anti-Glut4 (GT-41-A; Alpha diagnostic international), anti-Laminin2 (L0663; Sigma-Aldrich), anti-CD36 (80080; Abcam), anti-Dystrophin (15277; Abcam), and LysoTracker (ENZ-51005; Enzo).

Rong Y., Zhang S., Nandi N., Wu Z., Li L., Liu Y., Wei Y., Zhao Y., Yuan W., Zhou C., Xiao G., Levine B., Yan N., Mou S., Deng L., Tang Z., Liu X., Kramer H, & Zhong Q. (2022). STING controls energy stress-induced autophagy and energy metabolism via STX17. The Journal of Cell Biology, 221(7), e202202060.

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THP-1 Cell Surface Protein Analysis

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THP-1 cells were harvested, washed in PBS, and fixed with paraformaldehyde. With the exception of experiments to investigate lysosomal activity, no additional permeabilization was performed so as to allow the analysis of only the proteins expressed on the cell surface. To prevent nonspecific antibody binding, cells were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. After incubation with anti-TLR4 (sc-13593; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-Rac1 (03589; EMD Millipore, Darmstadt, Germany) antibody (dilution, 1:200) or LysoTracker (dilution, 1:1,000; ENZ-51005; Enzo Life Sciences, Farmingdale, NY, USA), THP-1 cells were stained with Alexa Fluor 488- or 594-conjugated secondary antibodies (dilution, 1:1,000; Enzo Life Sciences) and 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). The stained cells were observed under a Zeiss LSM800 confocal microscope (Carl Zeiss, Jena, Germany).

Kim J., Kim Y.J, & Kim J.W. (2018). Bacterial Clearance Is Enhanced by α2,3- and α2,6-Sialyllactose via Receptor-Mediated Endocytosis and Phagocytosis. Infection and Immunity, 87(1), e00694-18.

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Flow Cytometric Detection of P2Y6 Receptor

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To detect P2Y6 receptor on membrane surfaces by FACS, the cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) without permeabilization and were incubated and blocked using PBS containing 1% BSA for 30 min at room temperature. Next, the cells were labeled with the rabbit anti-P2Y6 receptor antibody (1:200; Alomone Labs) for 2 h at room temperature, followed by the Alexa Flour 488 goat anti-rabbit IgG antibody (Invitrogen) for 1 h at room temperature. Lysosomal activity was determined using Lyso-Tracker (Enzo Life Sciences) according to the manufacturer's protocol. The cells for flow cytometric analysis were washed three times with FACS buffer (PBS containing 1% FBS) and then analyzed using a FACSVerse flow cytometer (BD Biosciences). FlowJo software (Tree Star, OR, USA) was used for the data processing.

Shin S.H., Jeong J., Kim J.H., Sohn K.Y., Yoon S.Y, & Kim J.W. (2020). 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice. Frontiers in Immunology, 11, 710.

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Detecting P2Y6 Receptor Expression

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To detect the P2Y6 receptor on membrane surfaces, the cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) and were incubated and blocked using PBS containing 1% BSA for 30 min at room temperature. Next, the cells were labeled with the rabbit anti-P2Y6 receptor antibody (1:200, APR-011; Alomone Labs, Jerusalem, Israel) for 2 h at room temperature, followed by the Alexa Flour 488 goat anti-rabbit IgG antibody (Invitrogen) for 1 h at room temperature. Lysosomal activity was determined using Lyso-Tracker (ENZ-51005, Enzo Life Sciences) according to the manufacturer's protocol. To determine the intracellular trafficking of the P2Y6 receptor, THP-1 cells were co-stained with anti-P2Y6 receptor and anti-Rab5 antibody (Santa Cruz Biotechnology) for early endosome marker. Finally, the cells were washed with 1% fetal bovine serum/PBS and mounted with 4′,6-diamidino-2-phenylindole-containing fluorescence microscopy mounting medium (Invitrogen). The samples for confocal analysis were analyzed using a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).

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