Flow Cytometry Multiplexing Used to Analyze Metabolic ...€¦ · Jurkat (T lymphocyte cell type)...
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0.01 0.1 1 10 100 0 50 100 150 Amsacrine #110 (Fixable Live Dead Aqua) Amsacrine (μM) Normalized MFI 0.01 0.1 1 10 100 0 50 100 150 Benzyl Isothiocyanate #101 (Fixable Live Dead Aqua) BENZYL ISOTHIOCYANATE (μM) Normalized MFI 0.01 0.1 1 10 100 0 50 100 150 Benzyl Isothiocyanate #101 (Presto Blue) Benzyl Isothiocyanate (μM) Normalized RFU 0.01 0.1 1 10 100 0 50 100 150 Gambogic Acid Amide #7 (Fixable Live Dead Aqua) Gambogic Acid Amide (μM) Normalized MFI 0.01 0.1 1 10 100 0 50 100 150 Gambogic Acid Amide #7 (Presto Blue) Gambogic Acid Amide (μM) Normalized RFU 0.01 0.1 1 10 100 0 50 100 150 Gambogic Acid #2 (Fixable Live Dead Aqua) Gambogic Acid (μM) Normalized MFI 0.01 0.1 1 10 100 0 50 100 150 Gambogic Acid #2 (Presto Blue) Gambogic Acid (μM) Normalized RFU Leticia Montoya, Michelle Yan, Dan Beacham, Michael O’Grady, Marcy Wickett, April Anderson, Carolyn DeMarco, and Veronica Calderon Thermo Fisher Scientific, Willow Creek, Eugene, OR, USA, 97402 Screening Amsacrine (known chemotherapeutic for Leukemia) is a more potent drug for Jurkat cells than Ramos cells. ABSTRACT High throughput screening (HTS) is an extremely effective method for allowing researchers to identify putative active compounds for therapeutics. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry allows a researcher to study protein-protein interactions, metabolic activities, as well as DNA content in a single or multiplexed assay format. For this study, we screened the MicroSource Discovery System Killer Plates compound library and compared HTS using a plate reader with a flow cytometry live/dead assay. Jurkat (T lymphocyte cell type) and Ramos (B lymphocyte cell type) cells were used as cell models. Cells were cultured under standard conditions and either at hypoxic (1% using a Heracell™ VIOS 160i incubator) or hyperoxic (19%) oxygen levels. We also varied length of time the cells were treated with compound library (24, 48, or 72 hrs.). Membrane integrity and metabolic activity were measured as an output for evaluating cellular viability. To further assess cellular activity, post-screening analysis was implemented to establish EC50s of “hits” from compound library. Screening analysis identified target drugs that were further analyzed using cell health readouts with different concentrations of “hits” to assess compound toxicity. Results indicate that compound “hits” and potency differed in the screen depending on cell type, the mechanistic readout, and mode of readout used to perform the HTS experiments. INTRODUCTION High-throughput screening has become the industry standard for pharmaceutical drug screening and drug analysis. The ideal tool for investigating drug functions on cells is flow cytometry. Within this study we compared an acoustic flow cytometry platform with a fluorescent plate reader platform, and analyzed cell viability with different mechanisms of read-out. In combination with the different platforms used, we investigated the difference in oxygen levels and compound potency. The selection of promising compound leads is often poorly characterized due to the oxygen level difference in clusters of abnormal cell types within the human body. Jurkat (T lymphocyte cell type) and Ramos (B lymphocyte cell type) cells were used to study the affect of drugs on different types of white blood cells. White blood cells protect the body against foreign intruders; using these cells allows us to evaluate the effect of drugs tested and their involvement in cell-mediated immunity (T cells) and humoral immunity (B cells). Methods/Analysis – At 24, 48, and 72 hours post compound treatment cells were treated with either LIVE/DEAD TM Fixable Aqua Dead Cell Stain for 40 minutes or PrestoBlue TM Cell Viability Reagent for 30 minutes and analyzed on either the Attune TM NxT flow cytometer or the Varioskan TM Flash. CONCLUSIONS Analysis of cell health by screening can benefit from a multi-parametric approach, including different mechanistic and platform readouts. Flow cytometry and the use of multi-well plate auto samplers is an excellent tool for high throughput screening, and should be considered as a viable option for primary, secondary or tertiary compound screening. Oxygen concentrations need to be considered when performing screens and determining compound potency. Flow Cytometry Multiplexing Used to Analyze Metabolic Activity and Assess Pharmaceutical Compound Toxicity as A High Throughput Screening Tool Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com Figure 1. Screening of Ramos and Jurkat Cells Using PrestoBlue Cell Viability Reagent as the Read-out. Figure 2. Screening of Ramos and Jurkat Cells Using LIVE/DEAD Fixable Aqua Dead Cell Stain as the Read-out. Figure 7. Flow Analysis of Ramos cells Treated with Amsacrine Flow Analysis Cherry Picking Figure 3. Jurkat and Ramos Cells React Differently to the Same Drug 0.01 0.1 1 10 100 0 50 100 150 Clofazimine #175 (Fixable Live Dead Aqua) Clofazimine (μM) Normalized MFI 0.01 0.1 1 10 100 0 50 100 150 Amsacrine #110 (Presto Blue) Amsacrine (μM) Normalized RFU Figure 5. Similar Drugs Have Different Effects on Cell Type Gambogic Acid (natural product from Garcinia hanburyi shown to activate caspases and have anti- cancer activity) is a more potent drug than the amide version of same compound. Figure 6. Mechanistic Read-outs Give Two Different results Clofazimine (antibiotic used in multi-drug therapy for leprosy) is a more potent drug at 19% Oxygen for both Jurkat and Ramos cells when analyzed using cellular reduction potential as a cell health readout. Figure 4. Oxygen Levels have an Effect on Drug Potency Analyzing the redox potential of a cell gave a difference in compound potency compared to analyzing for membrane integrity. Cells plated at 40,000 cells per well, then treated with 10 μM of each drug from the Killer Collection from MicroSource Discovery Systems, Inc., and stored at either 19% Oxygen or 1% Oxygen in 5% CO 2 and at 37 ºC for 24, 48, and 72 hours. At for 24, 48, and 72 hours cells were treated with either LIVE/DEAD Fixable Aqua Dead Cell Stain or PrestoBlue Cell Viability Reagent and analyzed on either the Attune NxT or the Varioskan Flash. LIVE/DEAD Fixable Aqua Dead Cell Stain – Used to evaluate cell viability by flow cytometry. Cell viability was determined by the mean fluorescent intensity (MFI) of total cell population. Reactive dye permeates compromised cell membranes and reacts with free amines on the cell surface and the interior on the cell (Cell Membrane Integrity Read-out). ~Z’ = 0.8 PrestoBlue Cell Viability Reagent – Used to measure cytotoxicity and the proliferation of cells by a spectrometer. Cell viability was determined by measuring the mean fluorescent intensity of total cells per well and obtaining the relative fluorescent units (RFU) per well. Resazurin (PrestoBlue reagent) is reduced by the environment of living cells (Metabolic Read-out). ~Z’ = 0.7 Attune NxT Flow Cytometer – Designed to use acoustic-assisted hydrodynamic focusing, allowing for fast sample throughput rates. Varioskan Flash – Spectral scanning reader for fluorescence, absorbance, time resolved fluorescence, and luminescence. High fluorescence Low fluorescence 0.1 1 10 100 0 50 100 150 Clofazimine #175 (Presto Blue) Clofazimine (μM) Normalized RFU Jurkat O 2 19% Jurkat O 2 1% Ramos O 2 19% Ramos O2 1% Three distinct populations are seen with flow cytometry; live, dead, and unhealthy cells. Further analysis with a different readout method(s) are planned for the near future. 60 μM Amsacrine 0 μM Amsacrine 19 μM Amsacrine Jurkat O 2 19% Jurkat O 2 1% Ramos O 2 19% Ramos O2 1% Jurkat O 2 19% Jurkat O 2 1% Ramos O 2 19% Ramos O2 1% Jurkat O 2 19% Jurkat O 2 1% Ramos O 2 19% Ramos O2 1% Jurkat O 2 19% Jurkat O 2 1% Ramos O 2 19% Ramos O2 1% P3643 For Research Use Only. Not for use in diagnostic procedures Live Dead 19% 1% 19% 1% Amsacrine Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen Jurkat ~ 0.57 μM 0.26 μM 4.96 μM 6.60 μM Ramos ~ 23.30 μM ~ 20.17 μM ~ 71.57 μM ~ 64.95 μM PrestoBlue Cell Viability Reagent LIVE/DEAD Fixable Aqua Dead Cell Stain IC50 Drug Dose Response Table Clofazimine Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen Jurkat 10.69 μM ~ 30.86 μM NA NA Ramos 4.77 μM ~ 9.21 μM NA NA PrestoBlue Cell Viability Reagent LIVE/DEAD Fixable Aqua Dead Cell Stain IC50 Drug Dose Response Table Gambogic Acid Amide Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen Jurkat 0.60 μM 0.58 μM 2.50 μM 2.78 μM Ramos 0.19 μM 0.19 μM 1.01 μM 2.25 μM PrestoBlue Cell Viability Reagent LIVE/DEAD Fixable Aqua Dead Cell Stain IC50 Drug Dose Response Table Gambogic Acid Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen Jurkat 0.60 μM 0.58 μM ~ 0.57 μM ~ 0.53 μM Ramos 0.19 μM 0.19 μM ~ 0.30 μM ~ 0.33 μM IC50 Drug Dose Response Table LIVE/DEAD Fixable Aqua Dead Cell Stain PrestoBlue Cell Viability Reagent Benzyl Isothiocyanate Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen Jurkat 1.48 μM 1.06 μM NA NA Ramos 1.50 μM ~2.63 μM NA NA IC50 Drug Dose Response Table PrestoBlue Cell Viability Reagent LIVE/DEAD Fixable Aqua Dead Cell Stain © 2016 Thermo Fisher Scientific Inc. All rights reserved. All Thermo Fisher Scientific and its subsidiaries unless otherwise specified Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com 0 24 48 72 96 120 144 168 192 10 100 Jurkat 24 hours Fixable Live Dead Aqua Sample # % Dead Cells 2. 7. 101. 110. 175. 2. 7. 101. 110. 175. 0 24 48 72 96 120 144 168 192 1 10 100 Ramos 24 hours Fixable Live Dead Aqua Sample # % Dead Cells 2. 7. 101. 110. 175. 2. 7. 101. 110. 175. 0 24 48 72 96 120 144 168 192 1 10 100 Jurkat 24 hours PrestoBlue Sample # RFU 2. 7. 101. 110. 175. 2. 7. 101. 110. 175. 0 24 48 72 96 120 144 168 192 1 10 100 Ramos 24 hours PrestoBlue Sample # RFU 2. 7. 101. 110. 175. 2. 7. 101. 110. 175. MATERIALS AND METHODS
Transcript of Flow Cytometry Multiplexing Used to Analyze Metabolic ...€¦ · Jurkat (T lymphocyte cell type)...
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Leticia Montoya, Michelle Yan, Dan Beacham, Michael O’Grady, Marcy Wickett, April Anderson, Carolyn DeMarco, and Veronica Calderon Thermo Fisher Scientific, Willow Creek, Eugene, OR, USA, 97402
Screening
Amsacrine (known chemotherapeutic for
Leukemia) is a more potent drug for Jurkat
cells than Ramos cells.
Cherry Picking – Cells were treated with select drugs and stored at either 19% Oxygen or 1%
Oxygen in 5% CO2 and at 37 ºC for 24, 48, and 72 hours.
ABSTRACT
High throughput screening (HTS) is an extremely effective method for allowing
researchers to identify putative active compounds for therapeutics. Recently, flow
cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell
analysis. Flow cytometry allows a researcher to study protein-protein interactions,
metabolic activities, as well as DNA content in a single or multiplexed assay format. For
this study, we screened the MicroSource Discovery System Killer Plates compound library
and compared HTS using a plate reader with a flow cytometry live/dead assay.
Jurkat (T lymphocyte cell type) and Ramos (B lymphocyte cell type) cells were used as
cell models. Cells were cultured under standard conditions and either at hypoxic (1%
using a Heracell™ VIOS 160i incubator) or hyperoxic (19%) oxygen levels. We also
varied length of time the cells were treated with compound library (24, 48, or 72 hrs.).
Membrane integrity and metabolic activity were measured as an output for evaluating
cellular viability. To further assess cellular activity, post-screening analysis was
implemented to establish EC50s of “hits” from compound library.
Screening analysis identified target drugs that were further analyzed using cell health
readouts with different concentrations of “hits” to assess compound toxicity. Results
indicate that compound “hits” and potency differed in the screen depending on cell type,
the mechanistic readout, and mode of readout used to perform the HTS experiments.
INTRODUCTION
High-throughput screening has become the industry standard for pharmaceutical drug
screening and drug analysis. The ideal tool for investigating drug functions on cells is flow
cytometry. Within this study we compared an acoustic flow cytometry platform with a
fluorescent plate reader platform, and analyzed cell viability with different mechanisms of
read-out. In combination with the different platforms used, we investigated the difference
in oxygen levels and compound potency. The selection of promising compound leads is
often poorly characterized due to the oxygen level difference in clusters of abnormal cell
types within the human body.
Jurkat (T lymphocyte cell type) and Ramos (B lymphocyte cell type) cells were used to
study the affect of drugs on different types of white blood cells. White blood cells protect
the body against foreign intruders; using these cells allows us to evaluate the effect of
drugs tested and their involvement in cell-mediated immunity (T cells) and humoral
immunity (B cells).
Methods/Analysis – At 24, 48, and 72 hours post compound treatment cells were
treated with either LIVE/DEADTM Fixable Aqua Dead Cell Stain for 40 minutes or
PrestoBlueTM Cell Viability Reagent for 30 minutes and analyzed on either the AttuneTM
NxT flow cytometer or the VarioskanTM Flash.
CONCLUSIONS
Analysis of cell health by screening can benefit from a multi-parametric approach,
including different mechanistic and platform readouts.
Flow cytometry and the use of multi-well plate auto samplers is an excellent tool for high
throughput screening, and should be considered as a viable option for primary, secondary
or tertiary compound screening.
Oxygen concentrations need to be considered when performing screens and determining
compound potency.
Flow Cytometry Multiplexing Used to Analyze Metabolic Activity and
Assess Pharmaceutical Compound Toxicity as A High Throughput
Screening Tool
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
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1 0 9 .
1 1 0 .1 1 1 .
1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .
1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .
1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .1 5 3 .1 5 4 .1 5 5 .1 5 7 .
1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .
1 6 5 .
1 6 6 .
1 6 7 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .1 7 4 .
1 7 5 .1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 1 .
1 8 2 .
1 8 3 .
1 8 4 .
1 8 5 .1 8 6 .
1 8 7 .1 8 8 .
1 8 9 .
1 9 0 .
1 9 1 .
1 .
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .
1 2 .
1 3 .1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .5 9 .
6 1 .
6 2 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 6 .7 7 .7 8 .7 9 .8 0 .8 1 .8 2 .
8 3 .
8 5 .
8 6 .
8 7 .
8 8 .8 9 .
9 0 .
9 1 .
9 2 .9 3 .9 4 .
9 5 .
9 8 .
9 9 .1 0 0 .1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .
1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .1 2 3 .
1 2 4 .
1 2 5 .
1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .1 4 9 .
1 5 0 .
1 5 2 .1 5 3 .1 5 4 .1 5 5 .1 5 7 .1 5 8 .1 5 9 .
1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 5 .1 6 6 .
1 6 7 .
1 7 0 .1 7 1 .1 7 2 .
1 7 3 .1 7 4 .
1 7 5 .1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 6 .
1 8 7 .1 8 8 .1 8 9 .
1 9 0 .
1 9 1 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
J u r k a t 7 2 h o u r s
P re s to B lu e
S a m p le #
RF
U
1 .
2 .
3 .
4 .5 .
6 .
7 .
8 .
9 .
1 0 .1 1 .1 2 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .
2 1 .2 2 .
2 3 .
2 4 .
2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .
3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 4 .7 5 .
7 6 .
7 7 .7 8 .7 9 .8 0 .8 1 .
8 2 .
8 3 .
8 5 .
8 6 .
8 7 .
8 8 .
8 9 .9 0 .
9 1 .
9 2 .9 3 .
9 4 .
9 5 .
9 7 .
9 8 .9 9 .
1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 6 .
1 0 7 .1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .
1 1 2 .
1 1 4 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .1 2 3 .
1 2 4 .
1 2 5 .
1 2 6 .
1 3 1 .
1 3 2 .
1 3 3 .
1 3 8 .
1 3 9 .
1 4 1 .
1 4 2 .
1 4 3 .1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 5 .1 5 7 .1 5 9 .
1 6 0 .
1 6 1 .
1 6 4 .1 6 5 .
1 6 6 .1 6 7 .
1 6 9 .
1 7 0 .
1 7 2 .
1 7 3 .
1 7 5 .
1 7 6 .
1 7 8 .
1 7 9 .
1 8 1 .
1 8 2 .
1 8 3 .
1 8 4 .
1 8 6 .1 8 7 .
1 8 8 .
1 9 0 .
1 9 1 .
2 .
3 .
4 .
5 .
6 .
7 .9 .
1 0 .
1 1 .1 2 .
1 3 .1 4 .
1 5 .
1 6 .
1 7 .1 9 .
2 0 .
2 1 .2 2 .
2 3 .
2 4 .
2 5 .2 6 .
2 7 .
2 8 .
2 9 .
3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .5 9 .
6 2 .
6 4 .
6 7 .6 8 .
6 9 .
7 0 .
7 1 .
7 3 .7 4 .7 6 .7 7 .7 8 .7 9 .8 0 .8 1 .8 2 .
8 3 .
8 5 .
8 6 .
8 7 .
9 0 .
9 1 .
9 2 .9 3 .9 4 .
9 5 .
9 8 .
1 0 0 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .1 1 0 .1 1 1 .
1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .1 3 8 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .1 4 4 .
1 4 5 .
1 4 6 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 4 .1 5 5 .
1 5 9 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 5 .1 6 6 .
1 6 7 .
1 6 9 .1 7 0 .1 7 1 .
1 7 2 .1 7 3 .1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 1 .
1 8 2 .
1 8 5 .1 8 6 .
1 8 7 .1 8 8 .1 8 9 .
1 9 0 .
1 9 1 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
R a m o s 4 8 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
1 .
2 .3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .
1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .8 0 .8 1 .
8 2 .
8 3 .8 4 .
8 5 .
8 6 .
8 7 .8 8 .8 9 .
9 0 .
9 1 .
9 2 .9 3 .
9 4 .
9 5 .9 6 .
9 7 .
9 8 .
9 9 .
1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .1 0 8 .
1 0 9 .
1 1 0 .1 1 1 .
1 1 2 .
1 1 3 .1 1 4 .
1 1 5 .1 1 6 .
1 1 7 .
1 1 8 .
1 1 9 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .1 2 5 .1 2 6 .1 2 7 .1 2 8 .
1 2 9 .
1 3 0 .
1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .1 5 0 .1 5 1 .1 5 2 .1 5 3 .1 5 4 .
1 5 5 .
1 5 7 .
1 5 8 .
1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .
1 6 5 .
1 6 6 .
1 6 7 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .
1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 1 .
1 8 2 .
1 8 3 .
1 8 4 .
1 8 5 .
1 8 6 .
1 8 7 .1 8 8 .
1 8 9 .1 9 0 .1 9 1 .
1 .
2 .
3 .
4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .
3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .
8 0 .
8 1 .
8 2 .
8 3 .
8 5 .
8 6 .
8 7 .
8 8 .
8 9 .
9 0 .9 1 .
9 2 .
9 3 .
9 4 .
9 5 .
9 6 .9 7 .
9 8 .
9 9 .1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .1 0 5 .1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .1 1 1 .
1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .1 2 3 .
1 2 4 .
1 2 5 .
1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .
1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 5 .
1 4 6 .
1 4 7 .1 4 8 .1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .1 5 4 .
1 5 5 .
1 5 7 .1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .
1 6 6 .
1 6 7 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .1 8 6 .1 8 7 .1 8 8 .
1 8 9 .
1 9 0 .
1 9 1 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
R a m o s 7 2 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
1 .
2 .3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .2 6 .2 7 .
2 8 .
2 9 .
3 0 .
3 1 .3 2 .
3 3 .
3 4 .
3 5 .3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .6 8 .
6 9 .
7 0 .
7 1 .7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .8 0 .
8 1 .8 2 .
8 3 .8 4 .
8 5 .8 6 .8 7 .8 8 .8 9 .
9 0 .9 1 .
9 2 .9 3 .9 4 .
9 5 .9 6 .
9 7 .
9 8 .
9 9 .
1 0 0 .1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .1 0 8 .
1 0 9 .
1 1 0 .1 1 1 .
1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 2 .1 2 3 .
1 2 4 .1 2 5 .1 2 6 .1 2 7 .1 2 8 .
1 2 9 .
1 3 0 .1 3 1 .
1 3 2 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .1 4 1 .
1 4 2 .
1 4 3 .1 4 6 .
1 4 7 .1 4 8 .
1 4 9 .
1 5 0 .1 5 1 .1 5 2 .
1 5 3 .
1 5 4 .
1 5 5 .
1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .
1 6 5 .
1 6 6 .
1 6 7 .1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .
1 7 4 .
1 7 5 .1 7 6 .1 7 7 .
1 7 8 .
1 7 9 .1 8 2 .
1 8 3 .1 8 4 .
1 8 5 .
1 8 6 .1 8 7 .
1 8 8 .
1 8 9 .1 9 0 .1 9 1 .
1 .
2 .3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .2 4 .
2 5 .
2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .
6 6 .
6 7 .6 8 .
6 9 .
7 0 .
7 1 .
7 3 .
7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .
8 0 .
8 1 .8 2 .
8 3 .
8 5 .
8 6 .
8 7 .
8 8 .8 9 .
9 0 .9 1 .
9 2 .
9 3 .
9 4 .
9 5 .
9 7 .
9 8 .
9 9 .1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .1 0 5 .1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .1 1 1 .
1 1 2 .
1 1 3 .1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .
1 2 6 .
1 2 7 .1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .1 3 8 .
1 3 9 .1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .
1 5 5 .
1 5 8 .1 5 9 .
1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .
1 6 5 .
1 6 6 .
1 6 7 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .
1 7 4 .1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .1 8 2 .
1 8 3 .
1 8 4 .1 8 5 .
1 8 6 .1 8 7 .
1 8 8 .
1 8 9 .
1 9 0 .1 9 1 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1 0
1 0 0
J u r k a t 4 8 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
1 .
2 .
3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .8 8 .
8 9 .
9 0 .9 1 .
9 2 .9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .
1 0 0 .1 0 1 .1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .
1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .
1 6 6 .1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .1 7 4 .
1 7 5 .1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .
1 8 6 .
1 8 7 .1 8 8 .1 8 9 .
1 9 0 .
1 9 1 .
1 9 2 .
1 .
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .
2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .2 7 .
2 8 .
2 9 .
3 0 .3 1 .
3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .
7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .
8 8 .
8 9 .
9 0 .9 1 .
9 2 .
9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .1 6 6 .
1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .1 7 3 .1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .1 8 6 .1 8 7 .1 8 8 .
1 8 9 .
1 9 0 .1 9 1 .
1 9 2 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1 0
1 0 0
J u r k a t 7 2 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
1 .
2 .3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .
2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .6 8 .6 9 .
7 0 .
7 1 .
7 2 .
7 3 .
7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .8 8 .
8 9 .
9 0 .
9 1 .
9 2 .9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .
1 0 0 .1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .
1 2 6 .
1 2 7 .1 2 8 .
1 2 9 .
1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .
1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .
1 6 6 .
1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .
1 7 4 .
1 7 5 .1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .1 8 6 .1 8 7 .1 8 8 .1 8 9 .
1 9 0 .1 9 1 .
1 9 2 .
1 .
2 .3 .4 .
5 .
6 .
7 .
8 .
9 .
1 0 .1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .
2 1 .
2 2 .
2 3 .
2 5 .
2 6 .
2 7 .
2 8 .
2 9 .3 0 .3 1 .
3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .4 6 .
4 7 .
4 8 .
4 9 .
5 0 .5 1 .
5 2 .
5 3 .
5 4 .
5 5 .5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .
6 6 .
6 7 .
6 8 .6 9 .7 0 .
7 1 .
7 2 .
7 3 .
7 4 .
7 5 .
7 6 .
7 7 .7 8 .
7 9 .
8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .8 8 .
8 9 .9 0 .
9 1 .
9 2 .
9 3 .
9 4 .
9 5 .9 6 .
9 7 .
9 8 .
9 9 .1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .1 4 8 .1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .
1 5 8 .
1 5 9 .
1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .
1 6 6 .1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .1 7 3 .1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .1 8 6 .1 8 7 .
1 8 8 .
1 8 9 .
1 9 0 .1 9 1 .
1 9 2 .
Jurkat 19% O2
Figure 1. Screening of Ramos and Jurkat Cells Using PrestoBlue Cell Viability
Reagent as the Read-out.
Figure 2. Screening of Ramos and Jurkat Cells Using LIVE/DEAD Fixable Aqua
Dead Cell Stain as the Read-out.
Figure 7. Flow Analysis of Ramos cells Treated with Amsacrine
Flow Analysis Cherry Picking
Figure 3. Jurkat and Ramos Cells React Differently to the Same Drug
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
C lo fa z im in e # 1 7 5
(F ix a b le L iv e D e a d A q u a )
C lo fa z im in e (µ M )
No
rm
ali
ze
d M
FI
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m o s O 2 1 9 %
R a m o s O 2 1 %
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(P re s to B lu e )
A m s a c r in e (µ M )
No
rm
ali
ze
d R
FU
J u rk a t O 2 1 9 % A
J u rk a t O 2 1 % A
R a m o s O 2 1 9 % A
R a m o s O 2 1 % A
Figure 5. Similar Drugs Have Different Effects on Cell Type
Gambogic Acid (natural product from Garcinia hanburyi shown to activate caspases and have anti-
cancer activity) is a more potent drug than the amide version of same compound.
Figure 6. Mechanistic Read-outs Give Two Different results
Clofazimine (antibiotic used in multi-drug
therapy for leprosy) is a more potent drug
at 19% Oxygen for both Jurkat and Ramos
cells when analyzed using cellular
reduction potential as a cell health readout.
Figure 4. Oxygen Levels have an Effect on Drug Potency
Analyzing the redox potential of a cell
gave a difference in compound
potency compared to analyzing for
membrane integrity.
Cells plated at 40,000 cells per well, then treated with 10 µM of each drug from the Killer Collection from
MicroSource Discovery Systems, Inc., and stored at either 19% Oxygen or 1% Oxygen in 5% CO2 and at
37 ºC for 24, 48, and 72 hours. At for 24, 48, and 72 hours cells were treated with either LIVE/DEAD
Fixable Aqua Dead Cell Stain or PrestoBlue Cell Viability Reagent and analyzed on either the Attune NxT
or the Varioskan Flash.
LIVE/DEAD Fixable Aqua Dead Cell Stain –
Used to evaluate cell viability by flow
cytometry. Cell viability was determined by
the mean fluorescent intensity (MFI) of total
cell population. Reactive dye permeates
compromised cell membranes and reacts with
free amines on the cell surface and the
interior on the cell (Cell Membrane Integrity
Read-out). ~Z’ = 0.8
PrestoBlue Cell Viability Reagent – Used to
measure cytotoxicity and the proliferation of
cells by a spectrometer. Cell viability was
determined by measuring the mean
fluorescent intensity of total cells per well and
obtaining the relative fluorescent units (RFU)
per well. Resazurin (PrestoBlue reagent) is
reduced by the environment of living cells
(Metabolic Read-out). ~Z’ = 0.7
Attune NxT Flow Cytometer – Designed to use acoustic-assisted hydrodynamic focusing,
allowing for fast sample throughput rates.
Varioskan Flash – Spectral scanning reader for fluorescence, absorbance, time resolved
fluorescence, and luminescence.
High fluorescence Low fluorescence
0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
C lo fa z im in e # 1 7 5
(P re s to B lu e )
C lo fa z im in e (µ M )
No
rm
ali
ze
d R
FU
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m o s O 2 1 9 %
R a m o s O 2 1 %
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(F ix a b le L iv e D e a d A q u a )
A m s a c rin e (µ M )
No
rm
ali
ze
d M
FI
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m os O 2 1 9 %
R a m o s O 2 1 %
Three distinct populations are seen with flow cytometry; live, dead, and unhealthy cells. Further analysis
with a different readout method(s) are planned for the near future.
60
µM
Am
sa
cri
ne
0
µM
Am
sa
cri
ne
19
µM
Am
sa
cri
ne
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(F ix a b le L iv e D e a d A q u a )
A m s a c rin e (µ M )
No
rm
ali
ze
d M
FI
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m os O 2 1 9 %
R a m o s O 2 1 %
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(F ix a b le L iv e D e a d A q u a )
A m s a c rin e (µ M )
No
rm
ali
ze
d M
FI
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m os O 2 1 9 %
R a m o s O 2 1 %
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(F ix a b le L iv e D e a d A q u a )
A m s a c rin e (µ M )N
orm
ali
ze
d M
FI
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m os O 2 1 9 %
R a m o s O 2 1 %
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(F ix a b le L iv e D e a d A q u a )
A m s a c rin e (µ M )
No
rm
ali
ze
d M
FI
J u rk a t O 2 1 9 %
J u rk a t O 2 1 %
R a m os O 2 1 9 %
R a m o s O 2 1 %
P3643
For Research Use Only. Not for use in
diagnostic procedures
Live Dead
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1 0
1 0 0
J u r k a t 4 8 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
1 .
2 .
3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .8 8 .
8 9 .
9 0 .9 1 .
9 2 .9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .
1 0 0 .1 0 1 .1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .
1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .
1 6 6 .1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .1 7 4 .
1 7 5 .1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .
1 8 6 .
1 8 7 .1 8 8 .1 8 9 .
1 9 0 .
1 9 1 .
1 9 2 .
1 .
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .
2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .2 7 .
2 8 .
2 9 .
3 0 .3 1 .
3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .
7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .
8 8 .
8 9 .
9 0 .9 1 .
9 2 .
9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .1 6 6 .
1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .1 7 3 .1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .1 8 6 .1 8 7 .1 8 8 .
1 8 9 .
1 9 0 .1 9 1 .
1 9 2 .
1 9 %
1 %
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1 0
1 0 0
J u r k a t 4 8 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
1 .
2 .
3 .4 .5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .
2 7 .
2 8 .
2 9 .
3 0 .3 1 .3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .
3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .8 8 .
8 9 .
9 0 .9 1 .
9 2 .9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .
1 0 0 .1 0 1 .1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .
1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .
1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .
1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .
1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .
1 6 6 .1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .
1 7 3 .1 7 4 .
1 7 5 .1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .
1 8 6 .
1 8 7 .1 8 8 .1 8 9 .
1 9 0 .
1 9 1 .
1 9 2 .
1 .
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .1 2 .
1 3 .
1 4 .
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
2 0 .
2 1 .2 2 .
2 3 .
2 4 .
2 5 .
2 6 .2 7 .
2 8 .
2 9 .
3 0 .3 1 .
3 2 .
3 3 .
3 4 .
3 5 .
3 6 .
3 7 .3 8 .
3 9 .
4 0 .
4 1 .
4 2 .
4 3 .
4 4 .
4 5 .
4 6 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
5 3 .
5 4 .
5 5 .
5 6 .
5 7 .
5 8 .
5 9 .
6 0 .
6 1 .
6 2 .
6 3 .
6 4 .
6 5 .6 6 .
6 7 .
6 8 .
6 9 .
7 0 .
7 1 .
7 2 .
7 3 .7 4 .
7 5 .7 6 .
7 7 .7 8 .
7 9 .8 0 .
8 1 .
8 2 .
8 3 .
8 4 .
8 5 .
8 6 .
8 7 .
8 8 .
8 9 .
9 0 .9 1 .
9 2 .
9 3 .
9 4 .
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .1 0 0 .
1 0 1 .
1 0 2 .
1 0 3 .
1 0 4 .
1 0 5 .
1 0 6 .
1 0 7 .
1 0 8 .
1 0 9 .
1 1 0 .
1 1 1 .1 1 2 .
1 1 3 .
1 1 4 .
1 1 5 .
1 1 6 .1 1 7 .
1 1 8 .
1 1 9 .
1 2 0 .
1 2 1 .1 2 2 .
1 2 3 .
1 2 4 .
1 2 5 .1 2 6 .
1 2 7 .
1 2 8 .
1 2 9 .1 3 0 .1 3 1 .
1 3 2 .
1 3 3 .1 3 4 .
1 3 5 .
1 3 6 .
1 3 7 .
1 3 8 .1 3 9 .
1 4 0 .
1 4 1 .
1 4 2 .
1 4 3 .
1 4 4 .
1 4 5 .
1 4 6 .
1 4 7 .
1 4 8 .
1 4 9 .
1 5 0 .
1 5 1 .
1 5 2 .
1 5 3 .
1 5 4 .1 5 5 .
1 5 6 .
1 5 7 .1 5 8 .1 5 9 .1 6 0 .
1 6 1 .
1 6 2 .
1 6 3 .
1 6 4 .1 6 5 .1 6 6 .
1 6 7 .
1 6 8 .
1 6 9 .
1 7 0 .
1 7 1 .1 7 2 .1 7 3 .1 7 4 .
1 7 5 .
1 7 6 .
1 7 7 .
1 7 8 .
1 7 9 .
1 8 0 .
1 8 1 .
1 8 2 .
1 8 3 .1 8 4 .1 8 5 .1 8 6 .1 8 7 .1 8 8 .
1 8 9 .
1 9 0 .1 9 1 .
1 9 2 .
1 9 %
1 %
Amsacrine
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat ~ 0.57 µM 0.26 µM 4.96 µM 6.60 µM
Ramos ~ 23.30 µM ~ 20.17 µM ~ 71.57 µM ~ 64.95 µM
PrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
IC50 Drug Dose Response Table
Clofazimine
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 10.69 µM ~ 30.86 µM NA NA
Ramos 4.77 µM ~ 9.21 µM NA NA
PrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
IC50 Drug Dose Response Table
Gambogic
Acid Amide
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 0.60 µM 0.58 µM 2.50 µM 2.78 µM
Ramos 0.19 µM 0.19 µM 1.01 µM 2.25 µM
PrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
IC50 Drug Dose Response Table
Gambogic
Acid
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 0.60 µM 0.58 µM ~ 0.57 µM ~ 0.53 µM
Ramos 0.19 µM 0.19 µM ~ 0.30 µM ~ 0.33 µM
IC50 Drug Dose Response Table
LIVE/DEAD Fixable
Aqua Dead Cell Stain
PrestoBlue Cell
Viability Reagent
Benzyl
Isothiocyanate
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 1.48 µM 1.06 µM NA NA
Ramos 1.50 µM ~2.63 µM NA NA
IC50 Drug Dose Response TablePrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
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0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1 0
1 0 0
J u r k a t 2 4 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
2 . 7 .
1 0 1 .
1 1 0 .
1 7 5 .
2 .
7 .
1 0 1 .
1 1 0 .
1 7 5 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
R a m o s 2 4 h o u r s
F ix a b le L iv e D e a d A q u a
S a m p le #
% D
ea
d C
ell
s
2 .
7 .
1 0 1 .
1 1 0 .
1 7 5 .
2 .
7 .
1 0 1 .
1 1 0 .
1 7 5 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
J u r k a t 2 4 h o u r s
P re s to B lu e
S a m p le #
RF
U 2 . 7 .
1 0 1 .
1 1 0 . 1 7 5 .
2 . 7 .
1 0 1 .
1 1 0 .
1 7 5 .
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
R a m o s 2 4 h o u r s
P re s to B lu e
S a m p le #R
FU
2 . 7 .
1 0 1 .1 1 0 . 1 7 5 .
2 .
7 . 1 0 1 .
1 1 0 .
1 7 5 .
MATERIALS AND METHODS
