Keratinocytes were loaded with pHrodo Red AM (Molecular Probes) according to the manufacturer’s instructions. Briefly, pHrodo Red AM was diluted 10x in PowerLoad concentrate, and this mixture was diluted 100x in Live Cell Imaging Solution (LCIS, Invitrogen). Meanwhile, HaCaT cells were washed 1x with sterile LCIS, and were incubated with the diluted pHrodo dye for 30 minutes while covered at 37°C with 5% CO2. Excess dye was aspirated, and cells were washed 2x with sterile LCIS. For real-time live imaging experiments, cells were incubated with pre-warmed DMEM + 10% FBS and infected with GAS at an MOI of 0.01. This lower MOI was used to ensure that cells would not be overwhelmed with bacteria over the time course. Infections were imaged on the Nikon Eclipse Ti-E microscope described above, fitted with an environmental chamber set to maintain 37°C and 5% CO2. Images were taken in the DIC and pHrodo (excitation 555 nm, emission 580 nm, exposure 10 ms) every 10 minutes for 8 hours. Images were reconstructed into time-lapse videos using ImageJ. Experiments were performed at least in triplicate for each condition to confirm individual phenotypes, and representative videos are shown.
Phrodo red am
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PHrodo Red AM is a fluorescent pH indicator used in cell-based assays. It is designed to measure changes in intracellular pH in live cells. The PHrodo Red AM dye exhibits a pH-dependent fluorescence, allowing for the monitoring of pH levels within the cellular environment.
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Lab products found in correlation
Eclipse ti e microscope, by Nikon (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Intracellular ph calibration buffer kit, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Nunc lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Glass bottom 96 well plate, by Ibidi (1 mentions)
Phrodo green dextran, by Thermo Fisher Scientific (1 mentions)
Lysosensor yellow blue dnd 160, by Thermo Fisher Scientific (1 mentions)
A14291dj, by Thermo Fisher Scientific (1 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)
Percevalhr, by Thermo Fisher Scientific (1 mentions)
Percevalhr plasmid, by Addgene (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Hanks balanced salt solution (hbss), by Beckman Coulter (1 mentions)
Cytoflex lx, by Beckman Coulter (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
20 protocols using phrodo red am
1
Live Imaging of Bacterial Infection
2
Imaging Granulocyte Intracellular pH Dynamics
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Freshly isolated human peripheral blood granulocytes (106) in a
volume of 10 μl were carefully added to the bottom of Nunc
Lab-Tek II Chamber Slides (Thermo Scientific, Germany) filled with
CO2-saturated, pre-warmed 150 mM NaHCO3(5% FCS,
1 μg ml−1 propidium
iodide (Sigma, Germany),
1 μg ml−1 Hoechst
33342 (Molecular Probes, Netherlands)). Cells were incubated at
37 °C and monitored in parallel via fluorescence microscopy
in a time-lapse manner for up to 60 min at different magnifications
(× 20 and × 60). Photos were processed in Adobe Photoshop
CS5. For live-cell imaging of intracellular pH (pHrodo Red AM, Molecular
Probes), cells were cultured at 37 °C/5%
CO2 in an incubation chamber and subjected to isotonic HBSS
supplemented with 50 mM NaHCO3 and 1% BSA.
volume of 10 μl were carefully added to the bottom of Nunc
Lab-Tek II Chamber Slides (Thermo Scientific, Germany) filled with
CO2-saturated, pre-warmed 150 mM NaHCO3(5% FCS,
1 μg ml−1 propidium
iodide (Sigma, Germany),
1 μg ml−1 Hoechst
33342 (Molecular Probes, Netherlands)). Cells were incubated at
37 °C and monitored in parallel via fluorescence microscopy
in a time-lapse manner for up to 60 min at different magnifications
(× 20 and × 60). Photos were processed in Adobe Photoshop
CS5. For live-cell imaging of intracellular pH (pHrodo Red AM, Molecular
Probes), cells were cultured at 37 °C/5%
CO2 in an incubation chamber and subjected to isotonic HBSS
supplemented with 50 mM NaHCO3 and 1% BSA.
3
Fluorescent Probes for Oxidative Stress
4
Evaluating Cellular and Endosomal pH
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To evaluate cellular and endosomal pH by microscopy, 3 × 103 cells were plated in a glass bottom 96-well plate (ibidi GmbH). After 24 h, the cells were treated for 30 min; at 37°C with staining buffer (Phenol red-free DMEM containing 20mM HEPES) containing 1:2000 pHrodo Red AM (Invitrogen; P35372) and 50 μg/ml pHrodo Green Dextran (Invitrogen; P35368) prepared in AMEM. The cells were then washed, medium replaced with phenol red-free DMEM, and imaged with a confocal microscope (Zeiss; LSM 880). The fluorescence intensity was measured, and results expressed as mean fluorescence intensity. For assessment of cellular pH level by flow cytometry, the cells were plated in a 24-well glass bottom plate at the density of 30 × 103 cells/well, then treated with the staining buffer containing 40 μg/ml pHrodo Green Dextran for 30 min at 37°C. The cells were then washed once with warm staining buffer prior to collecting the cells to record the signal by BD FACS Diva on CANTOII and analyze it using FlowJoV10. In both cases, an increase in the pHrodo Green signal indicates a decrease in endosomal pH level.
5
Lysosome Staining with Fluorescent Probes
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Lysosome staining with the fluorescent acidotropic probe LysoSensor™ Yellow/Blue DND-160 and pHrodo red AM® (Life Technologies, L-7545 and P35372, respectively) was performed according to the manufacturer's recommendations. Briefly, HEK-293T cells were seeded in glass-bottom dishes, left to adhere, and exposed to autophagic stimuli for 16 h as described above. LysoSensor Yellow/Blue (stock 1 mM in DMSO, pKa 4.2) or pHrodo (pKa 6.5) was then added directly to the medium at the final concentration of 5 μM and incubated for 2 h at 37°C, 5% CO2. At the end of the incubation cells were directly transferred to the confocal facility without further manipulation. LysoSensor Yellow/Blue fluorescence was excited at 760 nm (2-photon) and collected in the range 400 to 490 nm and 510 to 600 nm (emission) for ratiometric calculation. pHrodo red AM was excited at 543 nm and emission fluorescence was collected in the range 620 to 700 nm. pH was determined based on a calibration curve prepared with the same probe dissolved in buffers of different pH.
6
Phagocytosis Assay of Apoptotic T Cells
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Jurkat T cells were cultured overnight at a density of 5 × 106/ml and treated with camptothecin (10 μM) for 16 h. Growing or apoptotic Jurkat T cells were washed with PBS and incubated with 5 μM/ml pHrodo Red AM (P353721, Life Technologies) for 30 min at 37°C. The pHrodo-labeled Jurkat T cells were washed with live cell imaging solution (A14291DJ, Life Technologies). BMDMs were cultured on 12-well plates (5 × 105/well) for 16 h before treatment with 0.5 μg/ml LPS for 6 h (18 (link)). For analysis of phagocytosis, pHrodo-labeled Jurkat T cells were co-incubated with BMDMs for 1.5–2 h, washed with cold PBS, fixed in 10% neutral-buffered formalin for 10 min, counterstained with DAPI to visualize the nuclei, and analyzed by confocal microscopy (Zeiss LSM 710).
7
Monitoring Cytosolic ATP/ADP Ratio
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Single cell cytosolic ATP/ADP ratio measurements were performed using the genetically encoded biosensor PercevalHR [32] and pHrodo Red AM, intracellular pH indicator (c: p35372, Life Technologies). Cells were co-transfected with NC or PPP1R1A siRNA (10 nM) and 1 μg PercevalHR plasmid (c:49083, Addgene). Cells were starved for 2 h in SAB low glucose and preincubated for 1 h with pHrodo Red AM (diluted 1/1000). PercevalHR fluorescence was recorded by laser ex./em. at 490/ 530 nm and normalized to the pHrodo Red AM fluorescence emission (560/585 nm) on a Zeiss LSM510 confocal microscope for 30 min/well (scan time of 7.86 s).
8
Cardiac Pericytosis Monitoring with pHrodo Red
9
Intracellular pH Measurement in T Cells
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The pHrodo Red AM fluorogenic probe (ThermoFisher) was used for pH measurement. First, human or mouse T cells were plated on 96 well plates at a 100,000 cell/well concentration and washed with HBSS (Gibco). Then, cells were loaded with pHrodo Red AM diluted in a PowerLoad concentrate according to manufacturer’s instructions and incubated at 37° for 30 min. Cells were washed with HBSS and then cultured in different pH media with indicated treatments for 6 h. pH measurements based on pHrodo Ex/Em were taken by flow cytometry in the PE-A channel in a Cytoflex Lx instrument (Beckman Coulter).
ThepHivalues were estimated from calibration curves plotted for each sample by using pHrodo Red AM-loadedT cells incubated in solutions containing 10 μM nigericin and valinomycin at different pH (4.5, 5.5, 6.5, and 7.5).
ThepHivalues were estimated from calibration curves plotted for each sample by using pHrodo Red AM-loadedT cells incubated in solutions containing 10 μM nigericin and valinomycin at different pH (4.5, 5.5, 6.5, and 7.5).
10
Intracellular pH Sensing in Tumor Samples
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