 Introduction to the topic

 Working Principle of Seahorse Analyzer

 Working of a Seahorse XF Analyzer
 Different Instrumentations
 Applications
 Alternate Analytical Techniques to Seahorse
 Introduction to the topic
 Working Principle of Seahorse Analyzer
 Working of a Seahorse XF Analyzer
 Different Instrumentations
 Applications
 Alternate Analytical Techniques to Seahorse
 Introduction to the topic
 Working Principle of Seahorse Analyzer
 Working of a Seahorse XF Analyzer
 Different Instrumentations
 Applications
 Alternate Analytical Techniques to Seahorse
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Seahorse extracellular flux (XF) Analyzer measures OCR and ECAR
 Manufactured and developed by Seahorse Bioscience
 OCR – Oxygen Consumption rate
 ECAR – Extracellular acidification rate
 OCR used for measuring Oxidative phosphorylation
 ECAR used to measure cell Glycolysis
 Seahorse Analyzer measures both OCR & ECAR simultaneously
 Mammalian cells generate ATP by mitochondrial and non-
mitochondrial metabolism
 Cancer cells use different strategies to meet energetic and
catabolic needs
 Seahorse Analyzer continuously measures the oxygen
concentration and proton flux in cell supernatant over time
 These measurements are converted in OCR and ECAR values and
enable direct quantification of mitochondrial respiration and
glycolysis
 Mammalian cells generate ATP by mitochondrial and non-
mitochondrial metabolism
 Cancer cells use different strategies to meet energetic and
catabolic needs
 Seahorse Analyzer continuously measures the oxygen
concentration and proton flux in cell supernatant over time
 These measurements are converted in OCR and ECAR values and
enable direct quantification of mitochondrial respiration and
glycolysis
 Mammalian cells generate ATP by mitochondrial and non-
mitochondrial metabolism
 Cancer cells use different strategies to meet energetic and
catabolic needs
 Seahorse Analyzer continuously measures the oxygen
concentration and proton flux in cell supernatant over time
 These measurements are converted in OCR and ECAR values and
enable direct quantification of mitochondrial respiration and
glycolysis
 Mammalian cells generate ATP by mitochondrial and non-
mitochondrial metabolism
 Cancer cells use different strategies to meet energetic and
catabolic needs
 Seahorse Analyzer continuously measures the oxygen
concentration and proton flux in cell supernatant over time
 These measurements are converted in OCR and ECAR values and
enable direct quantification of mitochondrial respiration and
glycolysis
 Mammalian cells generate ATP by mitochondrial and non-
mitochondrial metabolism
 Cancer cells use different strategies to meet energetic and
catabolic needs
 Seahorse Analyzer continuously measures the oxygen
concentration and proton flux in cell supernatant over time
 These measurements are converted in OCR and ECAR values and
enable direct quantification of mitochondrial respiration and
glycolysis
 OCR is measured before & after the addition of inhibitors
 Measuring the baseline cellular OCR or baseline respiration
 Measuring ATP-linked respiration and proton leak respiration –
Oligomycin
 Measuring the maximal respiratory capacity – FCCP
 Measuring non-mitochondrial respiration – Antimycin A and rotenone
 OCR is measured before & after the addition of inhibitors
 Measuring the baseline cellular OCR or baseline respiration
 Measuring ATP-linked respiration and proton leak respiration –
Oligomycin
 Measuring the maximal respiratory capacity – FCCP
 Measuring non-mitochondrial respiration – Antimycin A and rotenone
 OCR is measured before & after the addition of inhibitors
 Measuring the baseline cellular OCR or baseline respiration
 Measuring ATP-linked respiration and proton leak respiration –
Oligomycin
 Measuring the maximal respiratory capacity – FCCP
 Measuring non-mitochondrial respiration – Antimycin A and rotenone
 OCR is measured before & after the addition of inhibitors
 Measuring the baseline cellular OCR or baseline respiration
 Measuring ATP-linked respiration and proton leak respiration –
Oligomycin
 Measuring the maximal respiratory capacity – FCCP
 Measuring non-mitochondrial respiration – Antimycin A and rotenone
 OCR is measured before & after the addition of inhibitors
 Measuring the baseline cellular OCR or baseline respiration
 Measuring ATP-linked respiration and proton leak respiration –
Oligomycin
 Measuring the maximal respiratory capacity – FCCP
 Measuring non-mitochondrial respiration – Antimycin A and rotenone
 OCR is measured before & after the addition of inhibitors
 Measuring the baseline cellular OCR or baseline respiration
 Measuring ATP-linked respiration and proton leak respiration –
Oligomycin
 Measuring the maximal respiratory capacity – FCCP
 Measuring non-mitochondrial respiration – Antimycin A and rotenone
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 Glycolysis is determined by ECAR – lactic acid secretion/unit time
 Considering Lactic acid formed after conversion from pyruvate
 Release of additional metabolites from TCA cycle hinder analysis
 We cannot choose a Buffering agent like sodium bicarbonate
 Role of pH in changing the rate of glycolysis
 Using chemical inhibitors to increase the accuracy
 EACR is also measured before & after the addition of inhibitors
 Calculating the basal non-mitochondrial respiration using Glucose
 Calculating the glycolytic capacity using Oligomycin
 Using the glycolytic capacity to measure Glycolytic reserve
 Using an inhibitor 2-DG (2-Deoxy-D-glucose) to quantify metabolic
fluxes
 EACR is also measured before & after the addition of inhibitors
 Calculating the basal non-mitochondrial respiration using Glucose
 Calculating the glycolytic capacity using Oligomycin
 Using the glycolytic capacity to measure Glycolytic reserve
 Using an inhibitor 2-DG (2-Deoxy-D-glucose) to quantify metabolic
fluxes
 EACR is also measured before & after the addition of inhibitors
 Calculating the basal non-mitochondrial respiration using Glucose
 Calculating the glycolytic capacity using Oligomycin
 Using the glycolytic capacity to measure Glycolytic reserve
 Using an inhibitor 2-DG (2-Deoxy-D-glucose) to quantify metabolic
fluxes
 EACR is also measured before & after the addition of inhibitors
 Calculating the basal non-mitochondrial respiration using Glucose
 Calculating the glycolytic capacity using Oligomycin
 Using the glycolytic capacity to measure Glycolytic reserve
 Using an inhibitor 2-DG (2-Deoxy-D-glucose) to quantify metabolic
fluxes
 EACR is also measured before & after the addition of inhibitors
 Calculating the basal non-mitochondrial respiration using Glucose
 Calculating the glycolytic capacity using Oligomycin
 Using the glycolytic capacity to measure Glycolytic reserve
 Using an inhibitor 2-DG (2-Deoxy-D-glucose) to quantify metabolic
fluxes
 EACR is also measured before & after the addition of inhibitors
 Calculating the basal non-mitochondrial respiration using Glucose
 Calculating the glycolytic capacity using Oligomycin
 Using the glycolytic capacity to measure Glycolytic reserve
 Using an inhibitor 2-DG (2-Deoxy-D-glucose) to quantify metabolic
fluxes
● Real-time measurements of OCR and ECAR are made by isolating an
extremely small volume(about 2 microlitres)of medium above a
monolayer of cells within a plate.
● Cellular oxygen consumption (respiration) and proton excretion
(glycolysis) causes rapid, easily measurable changes to the
concentration of dissolved oxygen and free protons in the transient
microchambers.
● The assay is measured every 2-5 s by a solid state sensor residing 200
microns above the cell monolayer.
● Real-time measurements of OCR and ECAR are made by isolating an
extremely small volume(about 2 microlitres)of medium above a
monolayer of cells within a plate.
● Cellular oxygen consumption (respiration) and proton excretion
(glycolysis) causes rapid, easily measurable changes to the
concentration of dissolved oxygen and free protons in the transient
microchambers.
● The assay is measured every 2-5 s by a solid state sensor residing 200
microns above the cell monolayer.
● Real-time measurements of OCR and ECAR are made by isolating an
extremely small volume(about 2 microlitres)of medium above a
monolayer of cells within a plate.
● Cellular oxygen consumption (respiration) and proton excretion
(glycolysis) causes rapid, easily measurable changes to the
concentration of dissolved oxygen and free protons in the transient
microchambers.
● The assay is measured every 2-5 s by a solid state sensor residing 200
microns above the cell monolayer.
● Real-time measurements of OCR and ECAR are made by isolating an
extremely small volume(about 2 microlitres)of medium above a
monolayer of cells within a plate.
● Cellular oxygen consumption (respiration) and proton excretion
(glycolysis) causes rapid, easily measurable changes to the
concentration of dissolved oxygen and free protons in the transient
microchambers.
● The assay is measured every 2-5 s by a solid state sensor residing 200
microns above the cell monolayer.
● The instrument uses a sensor cartridge in a 24-well plate format with each
sensor equipped with two embedded fluorophores
● One of the fluorophores is quenched by oxygen and the other is sensitive to
change in pH.
● During the measurements the sensor is lowered to 200 µm above the cell
monolayer, forming a microchamber of about 2 µL
● The Seahorse Analyzer contains fibre optic bundles that emit light, exciting the
fluorophores and then measure the change in the fluorophore emission
● The instrument uses a sensor cartridge in a 24-well plate format with each
sensor equipped with two embedded fluorophores
● One of the fluorophores is quenched by oxygen and the other is sensitive to
change in pH.
● During the measurements the sensor is lowered to 200 µm above the cell
monolayer, forming a microchamber of about 2 µL
● The Seahorse Analyzer contains fibre optic bundles that emit light, exciting the
fluorophores and then measure the change in the fluorophore emission
● The instrument uses a sensor cartridge in a 24-well plate format with each
sensor equipped with two embedded fluorophores
● One of the fluorophores is quenched by oxygen and the other is sensitive to
change in pH.
● During the measurements the sensor is lowered to 200 µm above the cell
monolayer, forming a microchamber of about 2 µL
● The Seahorse Analyzer contains fibre optic bundles that emit light, exciting the
fluorophores and then measure the change in the fluorophore emission
● The instrument uses a sensor cartridge in a 24-well plate format with each
sensor equipped with two embedded fluorophores
● One of the fluorophores is quenched by oxygen and the other is sensitive to
change in pH.
● During the measurements the sensor is lowered to 200 µm above the cell
monolayer, forming a microchamber of about 2 µL
● The Seahorse Analyzer contains fibre optic bundles that emit light, exciting the
fluorophores and then measure the change in the fluorophore emission
● The instrument uses a sensor cartridge in a 24-well plate format with each
sensor equipped with two embedded fluorophores
● One of the fluorophores is quenched by oxygen and the other is sensitive to
change in pH.
● During the measurements the sensor is lowered to 200 µm above the cell
monolayer, forming a microchamber of about 2 µL
● The Seahorse Analyzer contains fibre optic bundles that emit light, exciting the
fluorophores and then measure the change in the fluorophore emission
● There are 4 injector ports per well
that can be used to add various
inhibitors, catalysts , substrates
etc.

● The inputs are defined by the

user.

● Oligomycin, FCCP, rotenone, 2-

DG are some of the commonly
used inhibitors.
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● Platform to simultaneously measure two parameters (OCR and ECAR)
● Rapid, real- time and label-free technology
● Microplate format and high throughput
● Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
● Up to four injection ports
● Sensitive and precision optical microsensor detection system
● Intuitive software with touch screen interface and data analysis system
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● XF cell culture microplate
● Fibre optic sensors
● H+ sensors
● O2 sensors
● Assay medium
● Drug delivery ports
● Islets.
● Cell monolayer
● Cancer
● Neurodegeneration
● Immunology
● Cardiovascular Diseases
● Mitochondrial Diseases
● Screening
● Obesity
● Diabetes
● Cancer
● Neurodegeneration
● Immunology
● Cardiovascular Diseases
● Mitochondrial Diseases
● Screening
● Obesity
● Diabetes
Cancer :
• Warburg’s Observations and Warburg effect
• Unification of metabolic pathways in aggressive tumour growth
• Metabolic reprogramming
• Enhanced glycolysis and energy production in cancerous cells
• Enhanced self sufficiency of growth
• Suppression of growth-suppressing enzymes
● Cancer
● Neurodegeneration
● Immunology
● Cardiovascular Diseases
● Mitochondrial Diseases
● Screening
● Obesity
● Diabetes
NEURODEGENERATION
Spare Capacity Reveals More Than Basal Metabolism

BASELINE OLIGO FCCP ROT / MYX

Sod2 +/+

Cell type:
Mouse CD-1
Synaptosomes
● Cancer
● Neurodegeneration
● Immunology
● Cardiovascular Diseases
● Mitochondrial Diseases
● Screening
● Obesity
● Diabetes
IMMUNOLOGY
Spare Capacity Affects Memory T Cell Development and Survival

Naïve T cells
Effector T
cells
Memory T
cells

Cell type:
Mouse Lymphocytes
Spleen & Lymph Nodes
Assay What it measures How it works Assay kits
Extracellular Oxygen O2 consumption rate As cell respiration lowers ab197243, ab197242
Consumption (OCR) Assay O2 concentration, dye
fluorescence increases

Intracellular Oxygen Assay Intra-cellular O2 levels Dye fluorescence is ab197245

inversely proportional to
oxygen concentration

Glycolysis Assay (ECAR) Extracellular acidification Lactate causes Core assay: ab197244
(glycolysis) rate extracellular acidification, Glycolysis stress test
dye fluorescence kits: ab222945 and ab2229
increases 46

Fatty Acid Oxidation Assay O2 consumption rate on As fatty acid oxidation Companion kit to OCR
blocking of sugar lowers O2 concentration, assay: ab217602
metabolism dye fluorescence Complete kit: ab222944
increases
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