Cell culture and differentiation
For 3T3-L1 preadipocytes, our differentiation protocol was based on previously described protocols29, 30
. In brief, we obtained 3T3-L1 preadipocytes from ATCC (CL-173, lot number 4715281), and propagated the cells in DMEM with 4g/l glucose (Hyclone) and 10% bovine calf serum supplement (BCS, Hyclone) in a 37 °C / 5% CO2 incubator. We regularly sub-cultured cells at 70% confluence and only used cells with less than six passages for differentiation. To initiate differentiation, we plated preadipocytes to a 100 mm culture dish at high density (3-4 millions in 20 ml medium) and incubated the cells overnight. On the next day (Day 0), we gently replaced the medium with stage I differentiation medium of DMEM: 4 g/l glucose supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products), 160 nM insulin (Sigma-Aldrich), 250 nM dexamethasone (Sigma-Aldrich), and 0.5 mM 3-isoobutyl-1-methylxanthine (Sigma-Aldrich). We renewed the medium once on Day 2. On Day 3, we switched the medium to stage II differentiation medium of DMEM with 10% FBS supplement and 160 nM of insulin. Starting on Day 5, we changed the medium to adipocyte maintenance medium of DMEM and 10% FBS supplement. We renewed the maintenance medium every other day until the cells were transferred into imaging plates.
For HL-60 neutrophil-like cells, we used previously described protocols to culture and differentiate the cells19
. In brief, cells were cultured in RPMI 1640 plus L-glutamine and 25 mM HEPES (Fisher Scientific) supplemented with antibiotic/antimycotic (Invitrogen) and 10% FBS (HyClone) in a 37 °C / 5% CO2 incubator. To induce differentiatiation, 1.3% of Dimethyl sulphoxide (DMSO) (Sigma-Aldrich) were added to cell culture at a density of 0.2 million cells/ml and maintained for 7 days.
For H460 lung cancer cells, we maintained the cells in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine and penicillin-streptomycin in a 37 °C / 5% CO2 incubator.
For 3T3-L1 preadipocytes, cells were harvested from culture dishes and transferred to 384-well imaging plates (Nalgene Nunc) pretreated with 0.01% sterile poly-L-Lysine solution (molecular weight 70,000-150,000, Sigma-Aldrich) 40 hr before fixation. After gentle aspiration of adipocyte maintenance medium, phosphate buffer saline (PBS) was applied to the dishes to rinse off residual FBS, which inhibited the trypsinization process. After trypsinization, we gently spun down the detached cells with centrifugation and resuspended them in low glucose adipocyte maintenance medium (DMEM with 1g/l glucose and 10% FBS supplement). The cell suspensions were well-mixed by gentle pipetting to reduce aggregations, and then immediately transferred to imaging plate using a multichannel pipette. The optimal cell density was ~7000 cells at 50 μl of medium per well. We centrifuged the plates at 200 rpm for 2 min to bring cells down to the bottom of the glass slides.
For HL-60 neutrophil-like cells, we coated 96-well imaging plates (Nalgene Nunc) with 30 μl of 100 μg/ml fibronection (BD Bioscience) diluted in distilled water for 1 hr at RT. Around 6000 differentiated HL-60 cells were plated into each well of the coated plate. After 20 min incubation at 37 °C, cell media was removed from each well, followed by addition of 30 μl chemoattractant solution, which consisted of 10 nM formyl met-leu-phe (fMLP, Sigma-Aldrich) diluted into Hanks’ balanced salt solution (HBSS, Invitrogen) with 1.2% of low endotoxin Bovine serum albumin (BSA, Sigma-Aldrich). Cells were then incubated at 37 °C for 2 min.
For H460 lung cancer cells, cells were plated at a density of 30,000 cells per well on 96-well (uncoated) imaging plates (Nalgene Nunc), and incubated at 37 °C overnight.
Cell fixation and immunofluorescence staining
For 3T3-L1 preadipocytes, to stain intracellular lipid droplets, we replaced the maintenance medium with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 493/503, Invitrogen) solution in DMEM at the concentration of 1 μg/l. After incubation for 30 min, we rinsed the BODIPY dye off with one wash of DMEM, and immediately fixed the cells with 4% para-formaldehyde (PFA, Electron Microscopy Sciences) in PBS for 15 min at RT. We kept the PFA solution at 37 °C and added it to the plate using an automatic microplate dispenser (Matrix WellMate, Thermo Scientific). At the end of incubation, the fixative was flicked out quickly, and quenching buffer of 50 mM Ammonium Chloride was immediately added to each well to stop the reaction of PFA. After 10 min, we gently rinsed the cell plate 3 times with Tris buffered Saline (TBS) using a plate washer (ELX405, BioTek).
We permeabilized fixed 3T3L1 cells with 0.2% Triton-X100 in TBS for 5 min and washed twice with TBS on ELX405. We added blocking solution of 5% BSA in TBST to each well. After 1 hr incubation, blocking solution was completely flicked out and replaced with primary antibody mixtures (one from mouse with one from rabbit). We used rabbit anti-HSL, anti-phospho-HSL (Ser565), anti-PPARγ, anti-C/EBPα (all from Cell Signaling Technology), anti-perilipin (Abcam), and mouse anti-adiponectin (gift from Dr. Philipp E. Scherer, UT Southwestern Medical Center). The plate was tightly sealed with Para film and incubated at 4 °C. After overnight incubation, we thoroughly rinsed off the primary antibodies with 3 washes of TBS and one wash of blocking buffer. Each wash had 10 min incubation time. The fixed cells were further incubated with AlexaFluor 647 conjugated anti-rabbit and AlexaFluor 546 conjugated anti-mouse antibodies (Invitrogen) for 1 hr, and washed 3 times with TBST. Lastly, we introduced 2 μg/ml Hoechst to the plate. After two wash of TBS, we preserved the plate in 0.1% freshly prepared Sodium Azide at 4 °C.
For HL-60 neutrophil-like cells, cells were fixed with 30 μl of 2x intracellular buffer (1.4 M KCl, 10 mM MgCl2, 20 mM EGTA, 200 mM Hepes pH 7.5; diluted from 10x with water), 640 mM sucrose and 8% PFA (Sigma) at RT for 15 min on each well. After a quick wash with 50 μl of TBS, we permeabilized cells with 50 μl of 0.2% Triton-X100 in TBS for 15 min at RT, and blocked the permeabilized cells with 50 μl of 3% BSA in TBST for 1 hr at RT. Then, cells were stained with primary antibody mixtures (one from mouse with one from rabbit). We used rabbit anti-Hem1 (gift from Dr. Orion Weiner, UCSF), anti-pPTEN (Biosource), anti-alpha tubulin, anti-Rac1/2/3, anti-pAkt (Thr308), or mouse anti-pMLC2 (all from Cell Signaling Technology). We diluted the primary antibodies in blocking buffer, and incubated cells with 30 μl of primary antibodies at 4 °C. After overnight incubation, we rinsed off the primary antibodies with 3 washes of 50 μl of 0.3% Triton-X100 in PBS for 5 min each. The fixed cells were further incubated with AlexaFluor-488 conjugated anti-mouse (Invitrogen), and AlexaFluor-546 conjugated anti-rabbit (Invitrogen) antibodies for 2 hr at RT, and washed 3 times with 50 μl of 0.3% Triton-X100 in PBS. Lastly, we incubated cells with 30 μl of AlexFluor-647 phalloidin (1:40 dilution from stock, Invitrogen) plus Hoechst (Invitrogen) for 30 min at RT. After three washes, cells were preserved in 50 μl of PBS with 0.1% Sodium Azide.
For H460 lung cancer cells, cells were analyzed for cell cycle phase using a bioimaging-certified cell-cycle kit (BD Biosience) according to the manufacturer’s protocol. Briefly, adherent cells were BrdU (Cell-cycle kit, BD Biosience) loaded at a concentration of 104 μM for 1 hr at 37 °C. At RT, cells were fixed with PFA for 15 min, permeabilized with methanol solution for 10 min, and blocked for 30 min with FBS. Cells were stored overnight in PBS at 4 °C. The next day, cells were DNase-treated (Cell-cycle kit, BD Biosience) for 1 hr at 37 °C, and stained with either rabbit anti-Cyclin A2 (Invitrogen), anti-Cyclin B1 (Santa Cruz), anti-p21-Waf1/Cip1, or anti-pRb (S608) (all from Cell Signaling Technology). These primary antibodies were diluted in FBS and incubated with cells for 2 hr at RT. Next, AlexaFluor-488 conjugated anti-BrdU, AlexaFluor-647 conjugated anti-pH3 (S28), Hoechst (all from Cell-cycle kit, BD Biosience), and either AlexaFluor-546 conjugated anti-rabbit (Invitrogen), AlexaFluor-555 phalloidin (Invitrogen), or AlexaFluor-555 β-tubulin (BD Bioscience), were diluted in FBS and incubated at RT for 2 hr with the cells. Cells were washed with PBS between steps.
Image acquisition and preprocessing
We acquired images using a 20x objectives on an inverted fluorescence microscope (TE-2000, Nikon), equipped with a 12-bit CCD camera (CoolSNAP HQ, Photometrics) and controlled by the Metamorph software (v7.1, Universal Imaging). Sixteen images were acquired for each imaging well, and saved as 1392×1040 16-bit TIFF files. Then, we subtracted background intensities from the images using the rolling ball algorithm in ImageJ software (v1.38l, NIH), and stitched the 16 images together using the TurboReg plugin for ImageJ (version Feb 14, 2007; http://bigwww.epfl.ch/thevenaz/turboreg
Data analysis software
All data analysis was performed using custom software written in Matlab v2007a (Mathworks), unless otherwise indicated. The software we used for automatic subpopulation identification, feature selection, and Virtual Molecular Profiles construction can be downloaded from http://www4.utsouthwestern.edu/altschulerwulab
Information on cell segmentation and categorization, phenotypic feature measurement and preprocessing, unsupervised subpopulation identification, supervised subpopulation classification, classification performance estimation, feature subset selection, Virtual Phenotypic Profiling clustering, noise-level measurement, population-averaged divergence measurement, and statistical analysis is available in Supplementary Methods