interactive cell analysis compressed
TRANSCRIPT
![Page 1: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/1.jpg)
Answer the questions with the leading cell
viability, cytoxicity and apoptosis assays.CLICK FOR MORE INFO
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![Page 2: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/2.jpg)
CellTiter-Glo®Assay
CellTiter 96® AQueous
One Solution
CellTiter-Blue®Viability Assay
CytoTox 96®Assay
CytoTox-ONE™Assay
LIVE CELLPROTEASE
DEAD CELLPROTEASE
DEAD CELLPROTEASE
LIVE CELLPROTEASE
CellTiter-Fluor™Viability Assay
CytoTox-Glo™ Assay
CytoTox-Fluor™ Assay
Cell Viability, Cytotoxicity and Apoptosis Assays
aCaspase-Glo® 9 Assay
Caspase-Glo® 8 Assay
Apo-ONE®Caspase 3/7 Assay
Caspase-Glo® 3/7 Assay
![Page 3: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/3.jpg)
Back to Start (Cell)
10Minutes
Add CellTiter-Glo® Reagent
ReadLuminescence
CellTiter-Glo®
Cell Viability AssayCat. # G7570, G7571,
G7572, G7573
Reagent contains Ultra-Glo® Luciferase,
luciferin, Mg2+, buffer, lysis reagent and ATPase
inhibitors
Sensitive to as few as 10 cells
ATPATP
ATP
ATPATP
ATPATP
ATP
ATPATP
Luciferin Oxyluciferin + LIGHT
Ultra-Glo®
Luciferase
ADP
aSee Data
![Page 4: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/4.jpg)
Back to Start (Cell)
10Minutes
Add CellTiter-Glo® Reagent
ReadLuminescence
CellTiter-Glo®
Cell Viability AssayCat. # G7570, G7571,
G7572, G7573
Reagent contains Ultra-Glo® Luciferase,
luciferin, Mg2+, buffer, lysis reagent and ATPase
inhibitors
Sensitive to as few as 10 cells
ATPATP
ATP
ATPATP
ATPATP
ATP
ATPATP
Luciferin Oxyluciferin + LIGHT
Ultra-Glo®
Luciferase
ADP
a
Two-fold serial dilution of Jurkat cells in a 384-well plate. Data points represent the mean and standard deviation of 8 replicates for each cell number.
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![Page 5: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/5.jpg)
1-4hr37°C
Add CellTiter 96® AQueous One Solution Reagent
ReadAbsorbance
490nm
CellTiter 96® AQueousOne Solution
Cell Proliferation AssayCat. # G3580, G3581, G3582
Cells are not lysed so if color development is not to your liking, put back in incubator.
MTS
MTSformazan(soluble)
PES reducedPES
NADH NAD+
PES transports reducing equivalents from the interior of the cell to the exterior.
Back to Start (Cell) aSee Data
![Page 6: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/6.jpg)
1-4hr37°C
Add CellTiter 96® AQueous One Solution Reagent
ReadAbsorbance
490nm
CellTiter 96® AQueousOne Solution
Cell Proliferation AssayCat. # G3580, G3581, G3582
Cells are not lysed so if color development is not to your liking, put back in incubator.
MTS
MTSformazan(soluble)
PES reducedPES
NADH NAD+
PES transports reducing equivalents from the interior of the cell to the exterior.
Back to Start (Cell) a
Effect of B9 hybridoma cell number on absorbance at 490nm measured using the CellTiter 96® AQueous One Solution Assay. Reagent reacted with cells for 1 hour prior to reading absorbance. Zero cell absorbance has not been substracted from the data.
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![Page 7: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/7.jpg)
1-4hr37°C
Add CellTiter-Blue Reagent
ReadFluorescence560Ex/590Em
CellTiter-Blue®Cell Viability Assay
Cat. # G8080, G8081, G8082
Cells are not lysed so if color development is not to your liking, put back in incubator. May need optimization.
Resazurin Resorufin
Resazurin is reduced by metabolically active cells. Dead cells cannot reduce the compound.
Reducing Enzymes
Back to Start (Cell) aSee Data
![Page 8: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/8.jpg)
CellTiter-Blue®Cell Viability Assay
Cat. # G8080, G8081, G8082
1-4hr37°C
Add CellTiter-Blue Reagent
ReadFluorescence560Ex/590Em
Cells are not lysed so if color development is not to your liking, put back in incubator. May need optimization.
Resazurin Resorufin
Resazurin is reduced by metabolically active cells. Dead cells cannot reduce the compound.
Reducing Enzymes
Back to Start (Cell) a
Relative ability of different cell types to reduce resazurin. Serial twofold dilutions of Jurkat or HepG2 cells were prepared at 100µl/well in a 96-well plate andcultured for 1.5 hours at 37°C. CellTiter-Blue® Reagent (20µl/well) was added and cells were incubated for 1 hour before recording fluorescence (560Ex/590Em)
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![Page 9: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/9.jpg)
Back to Start (Cell)
GF-AFC
GF + AFC
lcp
lcpThe peptide substrate Gly-Phe-AFC crosses the membrane and “Live Cell” Protease (lcp) within intact cells cleaves GF-AFC.
Add 100µl of CellTiter-Fluor™
Reagent
Read FluorescenceLive: 400Ex/505Em
0.5-3 hours
CellTiter-Fluor ™Cell Viability Assay
Cat. # G6080, G6081, and G6082
“Live Cell” Proteasedoes not functionoutside cell
aSee Data
![Page 10: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/10.jpg)
Back to Start (Cell)
GF-AFC
GF + AFC
lcp
lcpThe peptide substrate Gly-Phe-AFC crosses the membran and “Live Cell” Protease within intact cells cleaves GF-AFC.
Add 100µl of CellTiter-Fluor™
Reagent
Read FluorescenceLive: 400Ex/505Em
0.5-3 hours
CellTiter-Fluor ™Cell Viability Assay
Cat. # G6080, G6081, and G6082
“Live Cell” Proteasedoes not functionoutside cell
a
Serial dilutions of Jurkat cells were plated. Half received no treatment (viable) and the other half were treated to induce cytotoxicity (treated). CellTiter-Fluor reagent was added and incubated for 30 minutes at 37°C and fluorescence read (400Ex/505Em).
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![Page 11: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/11.jpg)
Back to Start (Cell)
LDHLD
H
INTINTFormazan
Lactate + NAD+ Pyruvate + NADH
Diaphorase
transfer 50µl of conditioned
culture media
Add 50µl of Substrate Mix
(mix just prior to use)
Read Absorbance at 490nm
30 minutes
CytoTox 96® Non-RadioactiveCytotoxicity Assay
Cat. # G1780
Add 50µl of Stop Solution
You can assay the cultured cells for viability, caspase activity, etc. after you remove the conditioned media for assay.
aSee Data
![Page 12: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/12.jpg)
Back to Start (Cell)
LDHLD
H
INTINTFormazan
Lactate + NAD+ Pyruvate + NADH
Diaphorase
transfer 50µl of conditioned
culture media
Add 50µl of Substrate Mix
(mix just prior to use)
Read Absorbance at 490nm
30 minutes
CytoTox 96® Non-RadioactiveCytotoxicity Assay
Cat. # G1780
Add 50µl of Stop Solution
You can assay the cultured cells for viability, caspase activity, etc. after you remove the conditioned media for assay.
a
Human HepG2 cells were plated at 40,000 cells per well and allowed to grow to confluency. Cells were treated for 24 hours with the indicated concentration of staurosporine before LDH release was measured with the CytoTox 96® Assay. Data were corrected for vehicle-only control values.
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![Page 13: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/13.jpg)
Back to Start (Cell)
LDHLD
H
ResazurinResorufin
Lactate + NAD+ Pyruvate + NADH
Diaphorase
Add 100µl of CytoTox-One™
Reagent
Read Fluorescence560Ex/590Em
10 minutes
CytoTox-ONE™Homogeneous Membrane Integrity Assay
Cat. # G7890, G7891, G7892
Add 50µlStop Solution
Also works if you take off a portion of the conditioned media to a new plate and perform the assay. Allows multiplexing.
aSee Data
![Page 14: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/14.jpg)
CytoTox-ONE™Homogeneous Membrane Integrity Assay
Cat. # G7890, G7891, G7892
Back to Start (Cell)
LDHLD
H
ResazurinResorufin
Lactate + NAD+ Pyruvate + NADH
Diaphorase
Add 100µl of CytoTox-One™
Reagent
Read Fluorescence560Ex/590Em
10 minutes
Add 50µlStop Solution
Also works if you take off a portion of the conditioned media to a new plate and perform the assay. Allows multiplexing.
a
Staurosporine dose response curve. Confluent HepG2 cells were treated for 24 hours with staurosporine from 48nM to 25μM to generate a dose response curve. %CVs were below 5% for all drug concentrations showing the robustness of both the CytoTox-ONE™ Assay and the robotic platform.
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![Page 15: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/15.jpg)
Back to Start (Cell)
dcpdcp
AAF-Luciferin AAF + Luciferin
Add 100µl of CytoTox-Glo™
Reagent
Read Luminescence
15 Minutes
CytoTox-Glo™ Cytotoxicity Assay
Cat. # G9290, G9291, and G9292
“dead cell” Proteasereleased from cells withcompromised membranes
UltraGlo®
Luciferase+
ATP
LIGHT
The Ala-Ala-Phe-Luciferincannot cross intact cell membranes
aSee Data
![Page 16: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/16.jpg)
Back to Start (Cell)
dcpdcp
AAF-Luciferin AAF + Luciferin
Add 100µl of CytoTox-Glo™
Reagent
Read Luminescence
15 Minutes
CytoTox-Glo™ Cytotoxicity Assay
Cat. # G9290, G9291, and G9292
“dead cell” Proteasereleased from cells withcompromised membranes
UltraGlo®
Luciferase+
ATP
LIGHT
The Ala-Ala-Phe-Luciferincannot cross intact cell membranes
a
Comparison of the CytoTox-Glo™ Assay to a fluorescent LDH assay. Jurkat cells were treated with digitonin at 30µg/ml to induce cytotoxicity.
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![Page 17: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/17.jpg)
Back to Start (Cell)
dcpdcp
AAF-Rhodamine110 AAF + Rhodamine 110
Add 100µl of CytoTox-Fluor™
Reagent
Read Fluorescence485Ex/520Em
0.5-3 hours
CytoTox-Fluor™Cytotoxicity Assay
Cat. # G9260, G9261, and G9262
“dead cell” Proteasereleased from cells withcompromised membranes
Can be multiplexed with Luminescent Caspase assays.
The Ala-Ala-Phe-RHO110cannot cross intact cell membranes
aSee Data
![Page 18: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/18.jpg)
Back to Start (Cell)
dcpdcp
AAF-Rhodamine110 AAF + Rhodamine 110
Add 100µl of CytoTox-Fluor™
Reagent
Read Fluorescence485Ex/520Em
0.5-3 hours
CytoTox-Fluor™Cytotoxicity Assay
Cat. # G9260, G9261, and G9262
“dead cell” Proteasereleased from cells withcompromised membranes
Can be multiplexed with Luminescent Caspase assays.
The Ala-Ala-Phe-RHO110cannot cross intact cell membranes
a
CytoTox-Fluor Assay multiplexed with Caspase-Glo® 3/7 Assay. LN-18 cells were plated at 10,000 cells/well and allowed to attach overnight. Cells were treated with staurosporine for 6 hours prior to assay. The CytoTox-Fluor Assay Reagent is added to wells and cytotoxicity measured after incubation for 30 minutes at 37°C. Caspase-Glo® 3/7 Reagent is added and luminescence measured after a 30-minute incubation.
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proCaspase 9Caspase 9
LEHD-Luciferin LEHD + Luciferin
Add 100µl of Caspase-Glo® 9
Reagent
Read Luminescence
0.5-3 hours
Caspase-Glo® 9 AssayCat. # G8210, G8211, G8212
UltraGlo®
Luciferase+
ATP
LIGHT
Back to Start (Cell)
• Reagent lyses cells and measures active Caspase 9 activity.•MG-132 inhibitor present to
inhibit caspase-like proteasome activity
aSee Data
![Page 20: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/20.jpg)
proCsp 9Csp 9
LEHD-Luciferin LEHD + Luciferin
Add 100µl of Caspase-Glo® 9
Reagent
Read Luminescence
0.5-3 hours
Caspase-Glo® 9 AssayCat. # G8210, G8211, G8212
UltraGlo®
Luciferase+
ATP
LIGHT
Back to Start (Cell)
• Reagent lyses cells and measures active Caspase 9 activity.•MG-132 inhibitor present to
inhibit caspase-like proteasome activity
a
Inclusion of proteasome inhibitor MG-132 improves Caspase-Glo 9 Assay data quality and confidence. Jurkat cells were plated at 25,000 cells per well in a 96 well plate. Apoptosis was induced for 3 hours with 5µM staurosporine or vehicle alone. Cells were assayed with Caspase-Glo 9 reagent with or without MG-132.
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![Page 21: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/21.jpg)
proCaspase 8Caspase 8
LETD-Luciferin LETD + Luciferin
Add 100µl of Caspase-Glo® 8
Reagent
Read Luminescence
0.5-3 hours
Caspase-Glo® 8 AssayCat. # G8200, G8201, G8202
UltraGlo®
Luciferase+
ATP
LIGHT
Back to Start (Cell)
• Reagent lyses cells and measures active Caspase 8 activity.•MG-132 inhibitor present to
inhibit caspase-like proteasome activity
aSee Data
![Page 22: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/22.jpg)
proCsp 8Csp 8
LETD-Luciferin LETD + Luciferin
Add 100µl of Caspase-Glo® 8
Reagent
Read Luminescence
0.5-3 hours
Caspase-Glo® 8 AssayCat. # G8200, G8201, G8202
UltraGlo®
Luciferase+
ATP
LIGHT
Back to Start (Cell)
• Reagent lyses cells and measures active Caspase 8 activity.•MG-132 inhibitor present to
inhibit caspase-like proteasome activity
a
Inclusion of proteasome inhibitor MG-132 improves Caspase-Glo 8 Assay data quality and confidence. U937 cells were plated at 15,000 cells per well in a 96 well plate. Apoptosis was induced for 5 hours with 100ng/well soluble recombinant TRAIL (TNF-related apoptosis-inducing ligand) or vehicle alone. Cells were assayed with Caspase-Glo 8 reagent with or without MG-132.
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proCaspase 3/7Caspase 3/7
DEVD-Luciferin DEVD + Luciferin
Add 100µl of Caspase-Glo® 3/7
Reagent
Read Luminescence
0.5-3 hours
Caspase-Glo® 3/7 AssayCat. # G8090, G8091,
G8092 and G8093
UltraGlo®
Luciferase+
ATP
LIGHT
Back to Start (Cell)
Reagent lyses cells and measures active
Caspase 3 or Caspase 7 activity
aSee Data
![Page 24: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/24.jpg)
proCsp 3/7Csp 3/7
DEVD-Luciferin DEVD + Luciferin
Add 100µl of Caspase-Glo® 3/7
Reagent
Read Luminescence
0.5-3 hours
Caspase-Glo® 3/7 AssayCat. # G8090, G8091,
G8092 and G8093
UltraGlo®
Luciferase+
ATP
LIGHT
Back to Start (Cell)
Reagent lyses cells and measures active
Caspase 3 or Caspase 7 activity
a
The Caspase-Glo® 3/7 Assay produces luminescence that is linear over a broad range of cell numbers. Jurkat cells were treated with anti-Fas mAb for 4.5 hours to induce apoptosis or were left untreated. Caspase-Glo® 3/7 Reagent was added directly to the cells in 96-well plates and incubated for 1 hour before recording luminescence. Each point represents the average of 4 wells. The "no cell" blank control value has been substracted from each.
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proCaspase 3/7Caspase 3/7
Rhodamine 110-(DEVD)2 2 DEVD + Rhodamine 110
Add 100µl of Apo-ONE
Reagent
0.5-18 hours
Apo-ONE® HomogeneousCaspase 3/7 Assay
Cat. # G7790, G7791, G7792
Back to Start (Cell)
Reagent lyses cells and measures active Caspase 3
or Caspase 7 activity
Read Fluorescence485Ex/520Em
aSee Data
![Page 26: Interactive Cell Analysis Compressed](https://reader034.vdocuments.mx/reader034/viewer/2022042714/556e323ed8b42a6a698b512e/html5/thumbnails/26.jpg)
proCsp 3/7Csp 3/7
Rhodamine 110-(DEVD)2 2 DEVD + Rhodamine 110
Add 100µl of Apo-ONE
Reagent
0.5-18 hours
Apo-ONE® HomogeneousCaspase 3/7 Assay
Cat. # G7790, G7791, G7792
Back to Start (Cell)
Reagent lyses cells and measures active Caspase 3
or Caspase 7 activity
Read Fluorescence485Ex/520Em
a
Increased sensitivity with Apo-ONE Caspase 3/7 Assay. Apoptosis was induced in Jurkat cells by 5 hour anti-Fas receptor antibody treatment.
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