PHAR240 Drug Technologies

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PHAR240 Drug Technologiesand New Drug TherapiesCytotoxicity Data AnalysisAnna Marie BabeyRm 308 McClymontPh. 6773 2333Email: [email protected]Experimental Plates• cultured cells seededinto each well of 96-well plate• organisation of platedeterminesorganisation oftreatment groups• create plate map thatmatches organisationPlate MapData Matches Plate Organisation• values = print-out from plate-reader• exactly matches plate = exactly matches plate map• create new Excel file• copy/paste values into first worksheet
0.838
0.853
0.846
0.870
0.880
0.882
0.866
0.868
0.872



0.857
0.856
0.866
0.872
0.880
0.872






0.836
0.797
0.821
0.410
0.441
0.433
0.821
0.797
0.805



0.705
0.727
0.719
0.355
0.341
0.425
0.797
0.837
0.845



0.619
0.680
0.697
0.885
0.845
0.853
0.789
0.830
0.814



0.580
0.596
0.619
0.845
0.861
0.853
0.790
0.853
0.781



0.533
0.557
0.541
0.829
0.869
0.853






0.449
0.495
0.488
0.837
0.805
0.829
0.077
0.073
0.080



Colour-Code for Clarity• will need to group all values for each extract together• plate organisation isn’t user-friendly for dataanalysis» makes mistakes more likely• will use location of each value to create spreadsheetfor each extract
0.854
0.848
0.852
0.884
0.878
0.889
0.879
0.881
0.889
0.000
0.000
0.000
0.862
0.869
0.866
0.884
0.878
0.888
0.000
0.000
0.000
0.000
0.000
0.000
0.820
0.804
0.828
0.445
0.437
0.476
0.836
0.829
0.812
0.000
0.000
0.000
0.750
0.758
0.752
0.367
0.422
0.390
0.804
0.796
0.836
0.000
0.000
0.000
0.678
0.655
0.686
0.877
0.893
0.869
0.852
0.836
0.820
0.000
0.000
0.000
0.632
0.655
0.624
0.861
0.869
0.836
0.828
0.820
0.788
0.000
0.000
0.000
0.562
0.546
0.610
0.873
0.861
0.884
0.000
0.000
0.000
0.000
0.000
0.000
0.476
0.468
0.523
0.828
0.836
0.827
0.064
0.063
0.068
0.000
0.000
0.000
Step 1: Separate Data by Extract• in this instance copy/paste can gowrong• use location instead:– Pt 1 is in cell A3therefore:=A3Raw DataPt 1 Pt 2 Pt 3
0.705
0.712
0.686
0.692
0.705
0.686
0.679
0.672
0.705
0.699
0.712
0.712
0.672
0.679
0.705
0.686
0.692
0.672
0.672
0.666
0.686
0.659
0.64
0.672
A B C D E F G H I J K L
0.854
0.848
0.852
0.884
0.878
0.889
0.879
0.881
0.889
0.000
0.000
0.000
0.862
0.869
0.866
0.884
0.878
0.888
0.000
0.000
0.000
0.000
0.000
0.000
0.820
0.804
0.828
0.445
0.437
0.476
0.836
0.829
0.812
0.000
0.000
0.000
0.750
0.758
0.752
0.367
0.422
0.390
0.804
0.796
0.836
0.000
0.000
0.000
0.678
0.655
0.686
0.877
0.893
0.869
0.852
0.836
0.820
0.000
0.000
0.000
0.632
0.655
0.624
0.861
0.869
0.836
0.828
0.820
0.788
0.000
0.000
0.000
0.562
0.546
0.610
0.873
0.861
0.884
0.000
0.000
0.000
0.000
0.000
0.000
0.476
0.468
0.523
0.828
0.836
0.827
0.064
0.063
0.068
0.000
0.000
0.000
123445678Step 2: Don’t Forget ConcentrationStep 2: Convert ng/well tolog Concentration• find well volume in assayinstructions (in Moodle)• convert ng/well to mg/ml• get Excel to do it for you• calculate log of concentrationfrom mg/ml• get Excel to do it for you• ensure these calculations arenext to raw datang/well
0.1
1
10
100
1000
10000
100000
1000000
RawDatang/well mg/ml log conc Pt 1 Pt 2 Pt 3Step 3: Calculate AverageBackground & Net Average MaximumStaining• background & maximum staining = plate-specific values• not transferable to other plates• need to evaluate whether or not presence of raw extract affectsmaximum staining• generally will use staining from Time 0 controls for averagemaximum• must subtract average background from average maximum
0.854
0.848
0.852
0.884
0.878
0.889
0.879
0.881
0.889
0.000
0.000
0.000
0.862
0.869
0.866
0.884
0.878
0.888
0.000
0.000
0.000
0.000
0.000
0.000
0.820
0.804
0.828
0.445
0.437
0.476
0.836
0.829
0.812
0.000
0.000
0.000
0.750
0.758
0.752
0.367
0.422
0.390
0.804
0.796
0.836
0.000
0.000
0.000
0.678
0.655
0.686
0.877
0.893
0.869
0.852
0.836
0.820
0.000
0.000
0.000
0.632
0.655
0.624
0.861
0.869
0.836
0.828
0.820
0.788
0.000
0.000
0.000
0.562
0.546
0.610
0.873
0.861
0.884
0.000
0.000
0.000
0.000
0.000
0.000
0.476
0.468
0.523
0.828
0.836
0.827
0.064
0.063
0.068
0.000
0.000
0.000
Step 4: Convert Raw Data• net values = raw data value – average background• fixed location for average background• % max staining = net value * 100/net average max• calculate average % maximum for each concentrationof extract
RawData
NetValues
% MaxStaining
Pt 1
Pt 2
Pt 3
Pt 1
Pt 2
Pt 3
Pt 1
Pt 2
Pt 3
Avg % Max
Step 5: Copy/Paste Is Your Friend• once calculations complete for 1 extract,move on to other 2 extracts from same plate• copy/paste first table to new location & repopulate:• insert new raw values, replace max value withone for that extract in calculations• organisation should suit you: vertical, horizontal,other
0.854
0.848
0.852
0.884
0.878
0.889
0.879
0.881
0.889
0.000
0.000
0.000
0.862
0.869
0.866
0.884
0.878
0.888
0.000
0.000
0.000
0.000
0.000
0.000
0.820
0.804
0.828
0.445
0.437
0.476
0.836
0.829
0.812
0.000
0.000
0.000
0.750
0.758
0.752
0.367
0.422
0.390
0.804
0.796
0.836
0.000
0.000
0.000
0.678
0.655
0.686
0.877
0.893
0.869
0.852
0.836
0.820
0.000
0.000
0.000
0.632
0.655
0.624
0.861
0.869
0.836
0.828
0.820
0.788
0.000
0.000
0.000
0.562
0.546
0.610
0.873
0.861
0.884
0.000
0.000
0.000
0.000
0.000
0.000
0.476
0.468
0.523
0.828
0.836
0.827
0.064
0.063
0.068
0.000
0.000
0.000
Step 6: New Plate/Same Approach• again, copy/paste isyour friend• do this for 3replicates per cellline across 3 celllinesCell Line p.30 – Extracts W-X-Y
0.723
0.727
0.726
0.748
0.746
0.740
0.751
0.749
0.744
0.741
0.750
0.751
0.731
0.725
0.728
0.745
0.747
0.744
0.738
0.744
0.746
0.764
0.737
0.703
0.705
0.712
0.686
0.672
0.666
0.686
0.737
0.717
0.703
0.717
0.710
0.730
0.692
0.705
0.686
0.659
0.640
0.672
0.717
0.737
0.703
0.697
0.737
0.751
0.679
0.672
0.705
0.697
0.703
0.730
0.670
0.683
0.656
0.737
0.738
0.683
0.699
0.712
0.712
0.683
0.737
0.690
0.656
0.697
0.663
0.731
0.703
0.724
0.672
0.679
0.705
0.730
0.724
0.690
0.737
0.751
0.730
0.737
0.758
0.723
0.686
0.692
0.672
0.676
0.683
0.724
0.717
0.737
0.737
0.064
0.066
0.071
EtOH 0.725Saline 0.745H2O 0.748t0 Ext W 0.728 Net t0 Ext W 0.661t0 Ext X 0.745 Net t0 Ext X 0.678t0 Ext Y 0.743 Net t0 Ext Y 0.676Bkgnd 0.067Ext WRawDataNetValues% MaxStainingng/well mg/ml log conc Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Avg % Max
0.1
0.0000005
-6.301
0.705
0.712
0.686
0.638
0.645
0.619
97
98
94
96
1
0.000005
-5.301
0.692
0.705
0.686
0.625
0.638
0.619
95
97
94
95
10
0.00005
-4.301
0.679
0.672
0.705
0.612
0.605
0.638
93
92
97
94
100
0.0005
-3.301
0.699
0.712
0.712
0.632
0.645
0.645
96
98
98
97
1000
0.005
-2.301
0.672
0.679
0.705
0.605
0.612
0.638
92
93
97
94
10000
0.05
-1.301
0.686
0.692
0.672
0.619
0.625
0.605
94
95
92
93
100000
0.5
-0.301
0.672
0.666
0.686
0.605
0.599
0.619
92
91
94
92
1000000
5
0.699
0.659
0.640
0.672
0.592
0.573
0.605
90
87
92
89
Ext XRawDataNetValues% MaxStainingng/well mg/ml log conc Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Avg % Max
0.1
0.0000005
-6.301
0.697
0.703
0.730
0.630
0.636
0.663
93
94
98
95
1
0.000005
-5.301
0.683
0.737
0.690
0.616
0.670
0.623
91
99
92
94
10
0.00005
-4.301
0.730
0.724
0.690
0.663
0.657
0.623
98
97
92
95
100
0.0005
-3.301
0.676
0.683
0.724
0.609
0.616
0.657
90
91
97
92
1000
0.005
-2.301
0.737
0.717
0.703
0.670
0.650
0.636
99
96
94
96
10000
0.05
-1.301
0.717
0.737
0.703
0.650
0.670
0.636
96
99
94
96
100000
0.5
-0.301
0.670
0.683
0.656
0.603
0.616
0.589
89
91
87
89
1000000
5
0.699
0.656
0.697
0.663
0.589
0.630
0.596
87
93
88
89
Ext YRawDataNetValues% MaxStainingng/well mg/ml log conc Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Avg % Max
0.1
0.0000005
-6.301
0.737
0.751
0.730
0.670
0.684
0.663
99
101
98
100
1
0.000005
-5.301
0.717
0.737
0.737
0.650
0.670
0.670
96
99
99
98
10
0.00005
-4.301
0.764
0.737
0.703
0.697
0.670
0.636
103
99
94
99
100
0.0005
-3.301
0.717
0.710
0.730
0.650
0.643
0.663
96
95
98
96
1000
0.005
-2.301
0.697
0.737
0.751
0.630
0.670
0.684
93
99
101
98
10000
0.05
-1.301
0.737
0.738
0.683
0.670
0.671
0.616
99
99
91
97
100000
0.5
-0.301
0.731
0.703
0.724
0.664
0.636
0.657
98
94
97
97
1000000
5
0.699
0.737
0.758
0.723
0.670
0.691
0.656
99
102
97
100

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