HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ICCB-Longwood/NSRB Screening Facility, Harvard Medical School 靶标：HCMV UL50

External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

HTRF-Ratio_Avg.NPI	HTRF-Ch1_A	HTRF-Ch2_A	HTRF-Ratio_A	HTRF-Ch1_B	HTRF-Ch2_B	HTRF-Ratio_B	HTRF-Ratio_Avg
2.3	17638	7444	23694	17895	7063	25336	24515
4.8	17347	7312	23724	17257	7071	24405	24064.5
4.2	17868	7517	23770	18158	7395	24554	24162
17.3	11844	7012	16891	13204	6392	20657	18774
6.6	12258	6577	18638	14321	6502	22026	20332
18.1	11561	6789	17029	13512	6666	20270	18649.5
-3.9	11113	5694	19517	11959	4942	24199	21858
10.3	12215	6757	18078	13988	6506	21500	19789
-20.7	10425	4592	22703	11876	4585	25902	24302.5
14.4	12356	6766	18262	13858	6888	20119	19190.5
5.8	12868	6545	19661	14017	6600	21238	20449.5
15.8	12205	6703	18208	13470	6818	19757	18982.5
3.2	11742	5785	20297	12927	6054	21353	20825
10.3	12241	6415	19082	13490	6587	20480	19781
-2.3	10948	5353	20452	12530	5499	22786	21619
9.1	12644	6696	18883	13920	6620	21027	19955
8.7	13443	7082	18982	14453	6863	21059	20020.5
-27	10044	4376	22952	11409	4149	27498	25225
10	11550	6340	18218	13279	6196	21432	19825
3.7	10765	5590	19258	12467	5606	22239	20748.5

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：11908 靶标：N/A

External ID: RMI-FANCM-MM2

Protocol: FP HTS. Screening took place at the University of Wisconsin Small Molecule Screening and Synthesis Facility. A master mix of RMI core complex and F-MM2 (30 microL per well) was plated in black 384 shallow well plates (ThermoFisher, Waltham, MA), using a BioTek "MicroFlo Select" reagent dispenser. Compounds were added using a Beckman FX liquid handler; 0.33 microL of 10 mM stock was added for a final compound concentration of 33 microM. MM2 and cMM2 were each added to 4 wells of master mix per plate to a final concentration of 10 muM to serve as controls. Following compound addition, plates were covered, centrifuged briefly, and incubated for 20 minutes at room temperature. FP measurements were taking using a Tecan "Safire 2" microplate reader. Instrument settings were as follows: top read, EX 470, EM 525/20, G-factor 0.89947. A suitable gain was calculated from the first plate of each day. Z' scores for were calculated for each plate; plates with Z' scores <0.5 were rerun prior to analysis.

Comment:

Concentration uM	Data readout
33	55
33	63
33	65
33	63
33	61
33	62
33	61
33	58
33	71
33	61
33	64
33	83
33	158
33	84
33	104
33	60
33	62
33	59
33	58
33	59

Concentration uM	Data readout
33	55
33	63
33	65
33	63
33	61
33	62
33	61
33	58
33	71
33	61
33	64
33	83
33	158
33	84
33	104
33	60
33	62
33	59
33	58
33	59

Concentration uM	Data readout
33	55
33	63
33	65
33	63
33	61
33	62
33	61
33	58
33	71
33	61
33	64
33	83
33	158
33	84
33	104
33	60
33	62
33	59
33	58
33	59

175277-72-2 靶点实验数据

Concentration uM	Data readout
33	55
33	63
33	65
33	63
33	61
33	62
33	61
33	58
33	71
33	61
33	64
33	83
33	158
33	84
33	104
33	60
33	62
33	59
33	58
33	59