eMI activity was measured in cells stably transduced with lentivirus containing the KFERQ-Split Venus reporter.27 (link),28 (link) Cells were plated in glass-bottom 96-well plates and eMI flux was assessed with the addition or not of endolysosomal protease inhibitors (20mM ammonium chloride and 100μM leupeptin; +N/L in text). Atter 16 hours, cells were fixed with 4% PFA and imaged using high-content microscopy (Operetta system, Perkin Elmer) set to collect images from ≥ 800 cells/condition. CMA activity was measured in cells stably transduced with lentivirus carrying the KFERQ-PS-Dendra reporter29 (link),30 (link) 16h hours after photoswitching with an LED lamp (405nm for 3 minutes), and macroautophagy activity in cells stably transduced with lentivirus containing the mCherry-GFP-LC3 reporter.69 (link) In both cases, cells were fixed with 4% PFA and imaged using the same high-content microscopy system as for the eMI reporter. Images were quantified using the manufacturer’s software to detect fluorescent puncta, and changes in eMI and CMA activity were quantified as changes in the number of fluorescent puncta per cell. Macroautophagy flux was determined by the conversion of mCherry+/GFP+ puncta (autophagosomes) into mCherry+ puncta only (autolysosomes) as a result of GFP quenching in the low pH of the lysosome. Nuclei were labeled with Hoechst.
Operetta system
Manufactured by PerkinElmer
Sourced in United States
The Operetta system is a high-content analysis platform designed for quantitative cellular imaging. It provides automated image acquisition, analysis, and data management capabilities to support advanced cellular research and screening applications.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Cellcarrier 96 well plate, by PerkinElmer (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Harmony harmony 4.1, by PerkinElmer (2 mentions)
Lsm 710, by Zeiss (2 mentions)
β catenin antibody, by BD (1 mentions)
Nativepage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Black 96 well plate, by Corning (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
C 12300, by PromoCell (1 mentions)
F3648, by Merck Group (1 mentions)
A 21245, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
C2456, by Merck Group (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Nuclight red virus, by Sartorius (1 mentions)
Biotracker atp red dye, by Merck Group (1 mentions)
29 protocols using operetta system
1
Quantifying Cellular Autophagy Pathways
2
Immunofluorescent Localization of β-Catenin
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Fixed cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% BSA, and stained with β-catenin antibody at 1:300 (BD Biosciences; Cat#610154) diluted in 2.5% BSA. After washing with phosphate-buffered saline with 0.1% Tween-20, cells were incubated in secondary antibody conjugated to 1:1,000 Alexa fluor (Invitrogen; Cat#A1103) diluted in 2.5% BSA for 2 h in the dark. Cells were washed in phosphate-buffered saline, 0.1% Tween-20, and DAPI was used to stain nuclei. Cells were imaged on Perkin Elmer Operetta System using a 20× air objective.
3
Measurement of Lysosomal CMA Activity
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CMA activity in vitro was measured using isolated intact lysosomes incubated with purified proteins at 37 °C in an isosmotic media (20 mm MOPS pH 7.3, 0.25 m sucrose) for 20 min. At the end of the incubation, lysosomes were collected by centrifugation and subjected to immunoblot (Kaushik & Cuervo, 2009 ). Binding was calculated as the amount of substrate protein bound to the lysosomal membrane in the absence of protease inhibitors and uptake by subtracting the amount of protein associated with lysosomes in the presence (protein bound to the lysosomal membrane and taken up by lysosomes) and absence (protein bound to the lysosomal membrane) of protease inhibitors. Where indicated, lysosomes with lower CMA activity were used in the incubation to determine nonselective binding of the purified proteins to cellular membranes.
CMA activity in intact cells was measured using lentivirus‐mediated expression of the KFERQ‐PS‐Dendra2 and high‐content microscopy (Koga et al.,2011 ). Cells were plated in 96‐well plate and photoactivated with a 405‐nm light‐emitting diode (LED; Norlux) for 4 min with the intensity of 3.5 mA (current constant). After 16 h, cells were fixed with 4% paraformaldehyde and images were captured with a high‐content microscope (Operetta system, Perkin Elmer) and quantification was performed with the manufacturer's software in a minimum of 800 cells (approx. nine fields).
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CMA activity in vitro was measured using isolated intact lysosomes incubated with purified proteins at 37 °C in an isosmotic media (20 m
CMA activity in intact cells was measured using lentivirus‐mediated expression of the KFERQ‐PS‐Dendra2 and high‐content microscopy (Koga et al.,
4
Quantifying Autophagy and CMA Activities
5
Microscopic Detection of S. aureus Phagosomal Escape
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Briefly, phagosomal escape of S. aureus expressing red fluorescent protein was microscopically detected in host cells stably expressing the fluorescent escape reporter YFP-CWT in the cytoplasm [42 (link),43 (link)]. The cell wall-targeting domain (CWT) of the metallopeptidase lysostaphin shows strong affinity for the bacterial cell wall and is efficiently recruited to S. aureus upon translocation of the pathogen to the host cytosol. S. aureus strains were transduced with a plasmid expressing mRFPmars [44 (link)] under the control of the constitutive SarAP1 promoter. Hence, phagosomal escape was evident by YFP-CWT recruitment to red-fluorescent bacteria. DNA was stained with DAPI or Hoechst 34,850. Images were recorded with an Operetta System (PerkinElmer) using a 20x objective. Image analysis was performed with Harmony (PerkinElmer). The software identified host cell cytoplasm, nuclei, and spots in either green (YFP-CWT; escape) or red channels (mRFP; S. aureus). The mean relative escape rates (number of escape events as a fraction of all intracellular S. aureus) were scored in biological triplicates and technical duplicates.
6
Measuring Chaperone-Mediated Autophagy Activity
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CMA activity in vitro was measured using isolated intact lysosomes incubated with purified proteins and subjected to immunoblot47 (link). Binding was calculated as the amount of substrate protein bound to the lysosomal membrane in the absence of protease inhibitors and uptake by subtracting the amount of protein associated with lysosomes in the presence (protein bound to the lysosomal membrane and taken up by lysosomes) and absence (protein bound to the lysosomal membrane) of protease inhibitors.
CMA activity in intact cells was measured using lentivirus-mediated expression of the KFERQ-PS-Dendra2 and high-content microscopy49 (link). Cells were plated in a 96-well plate and photoactivated with a 405 nm light-emitting diode (LED: Norlux) for 4 min with the intensity of 3.5 mA (current constant). After 16 h, cells were fixed with 4% paraformaldehyde, and images were captured with a high-content microscope (Operetta system, Perkin Elmer) and quantification was performed with the manufacturer’s software in a minimum of 800 cells (approx. 9 fields).
LAMP-2A dynamics. Multimerization of LAMP-2A at the lysosomal membrane was studied on 3–12% NativePAGE Bis-Tris Gels (Invitrogen) after solubilization in 1% octylglucoside (in 20 mM MOPS and 150 mM NaCl buffer)41 (link).
CMA activity in intact cells was measured using lentivirus-mediated expression of the KFERQ-PS-Dendra2 and high-content microscopy49 (link). Cells were plated in a 96-well plate and photoactivated with a 405 nm light-emitting diode (LED: Norlux) for 4 min with the intensity of 3.5 mA (current constant). After 16 h, cells were fixed with 4% paraformaldehyde, and images were captured with a high-content microscope (Operetta system, Perkin Elmer) and quantification was performed with the manufacturer’s software in a minimum of 800 cells (approx. 9 fields).
LAMP-2A dynamics. Multimerization of LAMP-2A at the lysosomal membrane was studied on 3–12% NativePAGE Bis-Tris Gels (Invitrogen) after solubilization in 1% octylglucoside (in 20 mM MOPS and 150 mM NaCl buffer)41 (link).
7
Quantifying Cell Proliferation with BrdU
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The Cellomics BrdU cell proliferation reagent kits (Thermo Scientific, Pittsburgh, PA, USA) was used for quantification of DNA replication per our previous publication [39 (link)]. As an alternative to 3H-thymidine, 5-bromo-2′-deoxyuridine (BrdU) enables us to detect DNA replication in actively proliferating cells using a monoclonal antibody against BrdU and DyLight 488 fluorophore-conjugated secondary antibody. Moreover, cell number and DNA content were quantified with DAPI staining. For image acquisition, Caki-2 and 786-O cells were fixed and analyzed using the high content screening Operetta system (Perkin-Elmer, Hamburg, Germany).
8
Visualizing P. cynomolgi Transcripts in Hepatocytes
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P. cynomolgi M infected primary rhesus hepatocytes cultured for 6 days in CellCarrier-96 well plates (Perkin-Elmer, Waltham, MA, USA) were fixed for 30 min. at RT in 4% paraformaldehyde in PBS (Affymetrix, Cleveland, Ohio, USA), dehydrated and stored at −20°C until further processing. RNA in situ detection was performed using the RNAscope Multiplex Kit (Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer’s instructions. RNAscope probes used were: gapdh (PcyM_1250000, region 113–997) and hsp70 (PcyM_0515400, region 606–1837). Following the RNA-FISH protocol, IFA was performed using rabbit anti-PcyHSP70 to stain the parasites as described above. Z-Stack images were acquired on the Operetta system (Perkin-Elmer) using a 40x objective NA 0.95 and maximum projections are shown.
9
Photoactivated KFERQ-PS-Dendra2 Fibroblasts
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Mouse fibroblasts transduced with lentivirus particles carrying KFERQ-PS-Dendra2 were plated in glass-bottom 96-well plates and photoactivated with a 405 nm light emitting diode (LED: Norlux) for 3 min with the intensity of 3.5 mA (current constant). After 16 h, cells were fixed with 4% paraformaldehyde and images were captured with a high-content microscope (Operetta system, Perkin Elmer) and quantification was performed with the manufacturer’s software in a minimum of 1,200 cells (approx. 9 fields).
10
Hypericin and Ampicillin Antibacterial Effects
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An overnight culture of P. aeruginosa (0.03 OD value, 100 µL) was incubated with hypericin (10 µM), ampicillin (100 µg/mL), or both (10 µM hypericin and 100 µg/mL ampicillin) for 3.5 h in a shaker at 180 rpm at room temperature in a 96-well flat black cell culture plate (Corning Incorporated, Corning, NY, USA). The treated bacterial cells were centrifuged at 20,810× g for 30 min. The medium was carefully removed from each well, 100 µL of DPBS was added, and fluorescence images were obtained. For assessing S. aureus hypericin intake, an overnight culture (0.05 OD value, 100 µL) was treated with 10 µM hypericin for 30 min at room temperature, after which 100 µL of sample was transferred to a black 96-well plate (Corning) and incubated for 1 h; 50 µL of the supernatant was removed, and fluorescence images were obtained. The fluorescence images were captured with an Operetta system (excitation wavelength range of 460–490 nm and emission wavelength range of 560–630 nm; PerkinElmer, Waltham, MA, USA) and the image software Harmony version 3.5. The images were later analyzed by ImageJ software (version 1.4, National Institutes of Health, Bethesda, MD, USA).
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