A purified preparation of X31 was labeled with a pH-sensitive fluorescent dye (pHrodo Green STP ester; ThermoFisher). Briefly, a 10 mM stock solution of the dye was made by adding 75 µL of DMSO to 500 µg pHrodo Green STP ester. Fifty micrograms of a purified preparation of X31 (1 mg per mL) was then added to the reconstituted dye and incubated at room temperature in the dark for 60 min. The labeled virus was then dialyzed against 1× PBS overnight at 4 °C in the dark. Eight micrograms of purified virus preparation were pre-incubated with 100 µg per mL of mAb for 30 min at RT and then were subsequently used to infect 6- to 8-week-old female BALB/c mice IN. Two hours after infection, mice were sacrificed, lungs were collected, and alveolar macrophages were analyzed by flow cytometry as described above. Internalized viral particles in alveolar macrophages were indicated with green fluorescence. Two controls were utilized for this experiment: (i) pHrodo-labeled virus and (ii) pHrodo-labeled virus with control IgG. The percentage of AMφ with internalized virus particles over total AMφ was calculated. The experiment was done in triplicates. One-way ANOVA and Dunnett’s multiple comparisons test were used to determine statistical significance (GraphPad Prism).
Phrodo green stp ester
Manufactured by Thermo Fisher Scientific
The PHrodo Green STP ester is a fluorogenic pH indicator that can be used to measure changes in pH within biological samples. It is designed to provide a fluorescent signal that is proportional to the local pH environment.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Fv1000, by Olympus (1 mentions)
Uplansapo 40 lens, by Olympus (1 mentions)
Gentlemacs c tube, by Miltenyi Biotec (1 mentions)
Adult brain dissociation kit, by Miltenyi Biotec (1 mentions)
Phrodo red succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Snarf 1 dextran, by Thermo Fisher Scientific (1 mentions)
Cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
15 protocols using phrodo green stp ester
1
Fluorescent Labeling of Influenza Virus
2
Labeling Dendritic Cells with pHrodo™ Green
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LAB strains conjugated with pHrodo™ Green STP ester (Thermo Fisher Scientific) were prepared from lyophilised LAB according to the manufacturer’s protocol. The purified pDCs and mDCs were cultured with 10 μg/mL of the pHrodoTM Green-conjugated LAB for 20 h. The cells were washed twice with FACS buffer and suspended in FACS buffer for analysis. The percentage of cells that took up the LAB was determined by flow cytometry analysis and the cells were qualitatively observed by confocal laser scanning microscopy (FV1000, Olympus, Tokyo, Japan) using a UPlanSApo 40× lens (Olympus, N.A. 0.95). For confocal laser scanning fluorescence imaging of both pDCs and mDCs, the pHrodo™ Green dye was excited with a 473-nm laser and the fluorescent emission between 500 and 600 nm was monitored. For immunocytochemical imaging, anti-CD11b-APC-Cy7 antibody was excited at 635 nm and the fluorescent emission between 655 and 755 nm was monitored, while anti-B220-APC was excited at 635 nm and the fluorescent emission between 655 and 755 nm was monitored.
3
Labeling Mycobacterium tuberculosis for MDM Infection
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Mtb was labelled with 100 µM pHrodo Green STP Ester (Thermo Fisher, Waltham, MA) in 100 mM sodium bicarbonate buffer, pH 8.5 for 30 min at room temperature, and then washed three times with phosphate buffered saline before proceeding to infection of MDMs. The DNA intercalating dye DRAQ7 was used at 3 µM in the same experiments to detect cell death.
4
Isolation and Labeling of Myelin
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Three hemispheres from PBS-perfused brains of two WT animals were put in one gentleMACS C-tube (Miltenyi Biotech, 130-093-237) and processed separately. According to the adult brain dissociation kit (Miltenyi Biotech, 130-107-677) the brain tissue was dissociated and the suspension was filtered through a 70 µm cell strainer and filled up to 10 ml with PBS. After centrifugation at 1500 rpm for 10 min at room temperature two times, the pellet was re-suspended in 3100 µl PBS, and a 900 µl debris removal solution was added. The solution was gently overlaid with cold PBS and centrifuged at 3000 x g for 10 min at 4 °C. The myelin layer was collected and washed with PBS. 500 μl of the myelin suspension were centrifuged at 3000 x g for 10 min at 4 °C and then re-suspended in a 0.1 M sodium bicarbonate buffer at 20 mg/ml. The pHrodo Green STP Ester (Thermo Fisher, P35369) was added at a final concentration of 100 µM to the myelin suspension. After 45 min of incubation, the myelin suspension was centrifuged at 13000 x g for 10 min at room temperature and three times washed with PBS. Finally, the labeled myelin was re-suspended in sterile PBS and stored at 4 °C. Unlabeled myelin was collected and stored at 4 °C.
5
Quantifying Phagocytic Uptake of Diverse Substrates
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Human α-synuclein preformed fibrils [26 ], myelin debris [27 ] and immunoglobulin G-opsonized red blood cells [28 (link)] were labelled with pHrodo™ Green STP ester or pHrodo™ Red succinimidyl ester (Thermo Fisher Scientific) as previously described and used at the following respective concentrations which were determined to be non-saturating: 1 μM, 15 μg/mL and 5 × 104 cells/mL. Bioparticles of pHrodo™ Green-labelled Escherichia coli (E. coli) were purchased from Thermo Fisher Scientific and used at a concentration of 25 μg/mL. Cells were incubated with the labelled substrates for three hours unless otherwise indicated, and before being counterstained with Hoechst 33342 (5 μg/mL). Total green fluorescence intensity per cell was quantified on a CellInsight CX5 High Content Screening Platform. All conditions were assessed in triplicate. Unchallenged cells were used to measure background/autofluorescence. Histograms were generated using the Python library Matplotlib. Internalization of fluorescein isothiocyanate (FITC) -labelled myelin was assessed similarly, except cells were washed with 0.4% trypan blue solution to quench extracellular fluorescence prior to imaging.
6
Intracellular pH Measurement in Dendritic Cells
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For the labeling of OVA with pHrodo, a stock solution of 9 mM pHrodo Green STP ester (Thermo Fisher) in DMSO was mixed with 10 volumes of 10 mg/ml OVA and incubated for 2 h at RT. DCs were pulsed with 1 mM pHrodo-conjugated OVA and 1 μg/ml LPS in the presence or absence of 500 μM α-tocopherol for 15 minutes, followed by a chase of 150 minutes. The fluorescence intensities of pHrodo was measured by flow cytometry at 20, 40, 60, 90, 120 and 150 minutes after starting with the chase (excitation: 488 nm; emission: 530/30 nm). pH was calibrated by measuring the fluorescence of pHrodo in a standard solution series of known pH.
For the SNARF-1 experiments, DCs adhered to glass bottom microdishes were washed and incubated for 30 minutes with 1 mg/ml SNARF-1 dextran 10,000 MW (D3303, Thermo Fisher) with or without 500 μM α-tocopherol or 1 μM PAO. DCs were imaged with a Leica SP8 confocal microscope and a 63 × 1.20 NA water immersion objective (540 nm excitation; 550–600 nm green and 620–700 nm red emission peaks of SNARF-1). The pH calibration curve was created by imaging SNARF-1 dextran-containing DCs that were incubated with buffers of different pH (10 mM phosphate buffer, 150 mM NaCl) and 0.003% Triton-X100.
For the SNARF-1 experiments, DCs adhered to glass bottom microdishes were washed and incubated for 30 minutes with 1 mg/ml SNARF-1 dextran 10,000 MW (D3303, Thermo Fisher) with or without 500 μM α-tocopherol or 1 μM PAO. DCs were imaged with a Leica SP8 confocal microscope and a 63 × 1.20 NA water immersion objective (540 nm excitation; 550–600 nm green and 620–700 nm red emission peaks of SNARF-1). The pH calibration curve was created by imaging SNARF-1 dextran-containing DCs that were incubated with buffers of different pH (10 mM phosphate buffer, 150 mM NaCl) and 0.003% Triton-X100.
7
Quantifying Myelin Debris Phagocytosis
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Human myelin debris were obtained and labelled with pHRodo Green STP Ester (Invitrogen) as previously described (Healy et al. [43 (link)]). Cells were plated in a black 96-well plate at equal density and challenged with 15 ng/mL of myelin debris. Following the incubation with myelin, cells were counterstained with Hoechst 33,342 (5 g/mL, Invitrogen) and green fluorescence intensity per cell was measured using a CellInsight CX7 High Content Screening Platform. All conditions were assessed in triplicate. GraphPad Prism 8.0 software was used for non linear regression of the kinetic curve, t50 estimation and t-test analysis. A p-value lower than 0.05 was considered statistically significant.
8
Engulfment Assay of Apoptotic Cells by Macrophages
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Engulfment was performed as previously described17 (link). Briefly, C57BL/6 J, 8-week-old female mouse was injected with 2 ml of 3% thioglycolate into the peritoneum. After 3 days, cells were collected from the mice peritoneum with DMEM containing 10% FBS. To induce apoptosis, PLB cells expressing SPOT-Xkr4-FLAG and XRCC4-tagRFP were treated with UV, and labeled with 0.1 μg/ml pHrodo Green STP ester (Invitrogen). Apoptotic cells were incubated with thioglycolate-elicited peritoneal macrophages at 37 °C for 4 h. Then, macrophages were detached by trypsin/EGTA (Nacalai) at 37 °C for 3 min. Detached macrophages were incubated with 400 fold-diluted APC-labeled anti-CD11b antibody (BioLgend) at 4 °C for 20 min in CHES buffer (20 mM CHES pH 9.0, 150 mM NaCl, and 2% dialyzed FBS). Finally, macrophages were washed and resuspended in analysis buffer (20 mM CHES pH 9.0, 150 mM NaCl) containing 0.25 μg/ml DAPI and analyzed by flow cytometry.
9
Flow Cytometry-based Cell Capture and Phagocytosis Assay
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For flow cytometry-based capture assays, target cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) from a CFSE cell proliferation kit (Invitrogen, C34554) for 5 min at room temperature, washed three times with PBS containing 5% FBS, and resuspended in RPMI with 5% FBS before cells (4 × 105) were added to MDMs and cocultured at 37°C. After 2 h of coculture, MDMs were extensively washed and analyzed by flow cytometry. Capture efficiency was determined as the percentage of CD11b+ cells containing CFSE-derived green fluorescence. For phagocytosis assays, target cells were labeled with 100 ng/ml pHrodo green STP ester (Invitrogen, P35369), pH 7.8, for 30 min at room temperature, resuspended in serum-free RPMI, and then added to MDMs. After 2 h of coculture at 37°C, MDMs were washed, collected, and analyzed. Phagocytosis efficiency was determined as the percentage of CD11b+ cells containing pHrodo-derived green fluorescence.
10
Myelin Isolation and Labeling for Phagocytosis
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Myelin was isolated from cerebral cortices of adult rat brains as described previously (Kotter et al., 2006 (link); Wu et al., 2021 (link)). The procedure consisted in mechanical homogenization of brain tissue in ice‐cold 0.3 M sucrose (T25 Digital Ultra‐Turrax; 40 ml/brain), ultracentrifugation on 0.83 M sucrose (75,000g, 4°C, 30 min), suspension of the interface in 20 mM Tris/HCl buffer, washing (12,000g, 4°C, 15 min) and resuspension in 5–10 ml buffer. Protein concentration was determined (Bio‐Rad protein assay) and the volume adjusted to 1 mg/ml protein. Aliquots of 0.5 ml were stored at −20°C until further processing.
For the analysis of phagocytosis with flow cytometry or a microplate reader, myelin was labeled with pHrodo Green STP Ester (Invitrogen, P35369). The dye was dissolved in dimethyl sulfoxide (DMSO) (500 μg/75 μl DMSO) and 1% added to myelin extract, which was previously washed and suspended in 0.5 ml of 0.1 M NaHCO3 (pH 8.4). After incubation for 45 min at RT, the labeled myelin was spun down (8 min, 12,000g) and the pellet resuspended (1 mg/ml) in DMEM medium without phenol red with 1% FBS.
For the analysis of phagocytosis with flow cytometry or a microplate reader, myelin was labeled with pHrodo Green STP Ester (Invitrogen, P35369). The dye was dissolved in dimethyl sulfoxide (DMSO) (500 μg/75 μl DMSO) and 1% added to myelin extract, which was previously washed and suspended in 0.5 ml of 0.1 M NaHCO3 (pH 8.4). After incubation for 45 min at RT, the labeled myelin was spun down (8 min, 12,000g) and the pellet resuspended (1 mg/ml) in DMEM medium without phenol red with 1% FBS.
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