Interactive Cell Analysis Compressed

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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

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

http://www.promega.com/catalog/catalogredirect.asp?partno=G7570


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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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


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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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


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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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



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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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



LDHLD

H

INTINTFormazan


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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LDHLD

H

ResazurinResorufin


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


CytoTox-ONE™Homogeneous Membrane Integrity Assay

Cat. # G7890, G7891, G7892


LDHLD

H

ResazurinResorufin


Diaphorase

Add 100µl of CytoTox-One™

Reagent


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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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



dcpdcp

AAF-Luciferin AAF + Luciferin

Add 100µl of CytoTox-Glo™

Reagent

Read Luminescence

15 Minutes

CytoTox-Glo™ Cytotoxicity Assay

Cat. # G9290, G9291, and G9292


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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dcpdcp

AAF-Rhodamine110 AAF + Rhodamine 110

Add 100µl of CytoTox-Fluor™

Reagent


0.5-3 hours

CytoTox-Fluor™Cytotoxicity Assay

Cat. # G9260, G9261, and G9262


Can be multiplexed with Luminescent Caspase assays.

The Ala-Ala-Phe-RHO110cannot cross intact cell membranes

aSee Data



dcpdcp

AAF-Rhodamine110 AAF + Rhodamine 110

Add 100µl of CytoTox-Fluor™

Reagent


0.5-3 hours

CytoTox-Fluor™Cytotoxicity Assay

Cat. # G9260, G9261, and G9262


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


• Reagent lyses cells and measures active Caspase 9 activity.•MG-132 inhibitor present to

inhibit caspase-like proteasome activity

aSee Data


proCsp 9Csp 9

LEHD-Luciferin LEHD + Luciferin


Reagent

Read Luminescence

0.5-3 hours


UltraGlo®

Luciferase+

ATP

LIGHT




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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proCaspase 8Caspase 8

LETD-Luciferin LETD + Luciferin


Reagent

Read Luminescence

0.5-3 hours


UltraGlo®

Luciferase+

ATP

LIGHT




aSee Data


proCsp 8Csp 8

LETD-Luciferin LETD + Luciferin


Reagent

Read Luminescence

0.5-3 hours


UltraGlo®

Luciferase+

ATP

LIGHT




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


Reagent lyses cells and measures active

Caspase 3 or Caspase 7 activity

aSee Data


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


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


Reagent lyses cells and measures active Caspase 3

or Caspase 7 activity


aSee Data


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


Reagent lyses cells and measures active Caspase 3

or Caspase 7 activity


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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Interactive Cell Analysis Compressed