High Throughput Imaging Assay for Hepatic Lipid Droplet Formation
Medical researchers world-wide now use the term "epidemic" or "pandemic" to describe the alarming incidence of obesity in modern society, which is estimated to occur in 30% of the general US population. Perhaps, most alarming is the high incidence of obesity in children and adolescents, which indicates that medical problems associated with obesity (e.g., diabetes, metabolic syndrome, and heart more ..
BioActive Compounds: 954
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH083261-01A1
Assay Provider: Dr. Patrick M. McDonough, Vala Sciences Inc.
Medical researchers world-wide now use the term "epidemic" or "pandemic" to describe the alarming incidence of obesity in modern society, which is estimated to occur in 30% of the general US population. Perhaps, most alarming is the high incidence of obesity in children and adolescents, which indicates that medical problems associated with obesity (e.g., diabetes, metabolic syndrome, and heart disease) will increase in the foreseeable future, as obesity often increases with age. The dominant cellular basis for obesity is increased accumulation of triglycerides in lipid droplets within the cell. The two major cell types in which lipid droplet formation leads to pathological problems are adipocytes and hepatocytes.
The overall goal of this project is to identify chemical probes for inhibiting lipid droplet formation in hepatocytes. Identification of such compounds will better our understanding of the biochemical/molecular pathways that lead to the development of fatty liver disease, and may identify lead compounds for consideration for therapeutic development.
Wu JC, Merlino G, Fausto N. Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha. Proc Natl Acad Sci U S A. Jan 18 1994;91(2):674-678.
de Alwis NM, Day CP. Non-alcoholic fatty liver disease: the mist gradually clears. J Hepatol. 2008;48 Suppl 1:S104-112.
Guo Y, Walther TC, Rao M, et al. Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature. May 29 2008;453(7195):657-661.
Beller M, Sztalryd C, Southall N, et al. COPI complex is a regulator of lipid homeostasis. PLoS Biol. Nov 25 2008;6(11):e292.
Szymanski KM, Binns D, Bartz R, et al. The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology. Proc Natl Acad Sci U S A. Dec 26 2007;104(52):20890-20895.
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) AML12 cells (mouse hepatocytes) were obtained from the ATCC
3) Culture Media: phenol-red free DMEM with L-glutamine, Pen-strep, and 10% Fetal Bovine Serum.
4) Oleic Acid Working Solution: water soluble Oleic Acid (Sigma, O1257, 5mM stock in PBS) diluted to 1mM in PBS.
5) Positive Control Working Solution: Triacsin-C (Biomol, E1218, 5mM stock in DMSO) diluted to 77.1uM in PBS. Additional DMSO is added to achieve a DMSO concentration of 2%.
6) Negative Control Working Solution: 2% DMSO in water.
7) Fixative Working Solution: 6% Paraformaldehyde (PFA) in PBS.
8) Permeabilization Buffer Working Solution: 0.1% BSA (Sigma, A7888) and 0.01% Saponin (Sigma, 84510) in PBS.
9) Lipid Stain Working Solution: BODIPY 493/503 (Invitrogen, D3922) diluted to 5ug/mL in PBS.
10) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).
1) 45ul of cell suspension (220,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
2) Incubate plates overnight or approx. 20 hours at 37 degrees C and 5% CO2.
3) 3.5ul of 100uM compound solution was added to columns 3 through 24 of the assay plates for a final assay compound concentration of 6.5uM and 0.13% DMSO. Compound addition was done on a Biomek FX with 384-head dispenser (Beckman).
4) 3.5ul of negative control (2% DMSO) working solution was added to column 2 using the Biomek FX with 384-head dispenser for a final assay concentration of 0.13% DMSO.
5) 3.5ul of the positive control (77.1uM Triacsin-C) working solution was added to column 1 of each plate manually for a final assay concentration of 5uM Triacsin-C and 0.13% DMSO.
6) 5.5ul of oleic acid working solution was added to each well using the Biomek FX with 384-head dispenser for a final assay oleic acid concentration of 100uM.
7) Plates were incubated overnight at 37 degree C and 5% CO2.
8) Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.
9) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA and plates were incubated for 40 minutes at room temperature.
10) Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
11) 40ul of permeabilization buffer working solution was added to each well using the Wellmate bulk dispenser and plates were incubated for 15 minutes at room temperature.
12) Permeabilization buffer was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
13) 40ul of lipid stain working solution was added to each well using the Wellmate bulk dispenser (Matrix) for a final concentration of 3.3 ug/ml and plates were incubated for 1 hour at room temperature.
14) Stain solution was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
15) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml and plates were sealed.
HCS System Settings and Image Analysis Protocol:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
- 20x 0.45 NA air objective
- Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
- 2 channels acquired sequentially: Exp1Cam1 = Bodipy 493/503 (lipid droplets) using 488 nm laser excitation and 540/70 emisssion filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 emission filters
- 2 fields per well
2) Image analysis was performed using the CyteSeer software (Vala Sciences) with the "Lipid Droplets" algorithm where "Nucleus" represents DAPI-stained nuclei and "Lipid" represents Bodipy 493/503 stained lipid droplets. The following CyteSeer software settings were used:
CHANNEL 1: "Nucleus"
- Nucleus Enabled: TRUE
- Nucleus Folder Name: Exp2Cam4
- Nucleus Thresholding Method: Savitsky-Golay
- Nucleus Sensitivity: 500.00%
- Nucleus Filtering Method: None
- Nucleus Filtering Radius: 1
- Nucleus Minimum Size: 5
CHANNEL 2: "Lipid"
- Lipid Enabled: TRUE
- Lipid Folder Name: Exp1Cam1
- Lipid Thresholding Method: Otsu
- Lipid Sensitivity: 100.00%
- Lipid Filtering Method: None
- Lipid Filtering Radius: 1
- Lipid Minimum Size: 1
3) Well averages of cell-by-cell metrics calculated from...
- NUCLEI IMAGES: cell count ("CellCount"), nuclear area ("AreaNm"), integrated intensity of the nucleus ("TIINiNm"),
- CELL (LIPID DOPLET) IMAGES: area of the lipid droplet mask ("AreaLm"=raw data, "AREALm_HMF"=Hybrid-Median-Filter corrected for systemic plate effects), integrated intensity of the lipid droplets ("TIILiLm"=raw data, "TIILiLm_HMF"= Hybrid-Median-Filter corrected for systemic plate effects),
4) Actives were determined using CBIS software (ChemInnovations) by calculating the % inhibition of the "TIILiLm_HMF" metric and using a hit criteria of %inhibition >50%. Wells with cell counts <300 in the 2 acquired images were flagged cytotoxic/low cell count. All flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".
Compounds with a %Inhibition >= 50% at 6. 5 uM concentration and greater than 500 cells are defined as actives in this assay. Wells with cell counts <300 in the 2 acquired images were flagged cytotoxic/low cell count. All flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % displacement in the assay demonstrated by a compound at 6.5 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
** Test Concentration.
Data Table (Concise)