[pdf] in cell analyzer 3000 high throughput multiplexed cellular toxicity

High Throughput Multiplexed Cellular Toxicity
*Jan Turner, Samantha Murphy, Elaine Adie, Angela Williams, Molly Price-Jones.
Amersham Biosciences Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, England., email: jan.turner@uk.amershambiosciences.com. Alamar Blue was obtained from Serotec , UK, cells were plus the cell count were obtained from each well as an Conventional cellular toxicological assays are based on incubated for 4 hours and fluorescence was measured homogenous assay. The Alamar Blue results showed the microscopic observation of toxic end points in fixed no significant change with dose (Fig. 2).
cells and for these reasons, are inherently limited both in terms of data throughput and content. Using Following the exposure period, cells were aspirated and commercially available apoptotic markers and the IN washed briefly with 100µl warmed phosphate buffered Cell Analyzer 3000, we have demonstrated and saline (PBS). This was followed by incubation with quantified multiple sub-toxic responses in individual 100µl of probe mixture for a total of 15 min 37 C before cells in both live and fixed heterogeneous primary cell being read using the IN Cell Analyzer 3000 (Fig. 1 and cultures and immortalized cell lines, while processing up to 40,000 data points in one plate. Pre-apoptotic perturbances have been simultaneously quantitated in Following Staurosporine exposure, Hoechst 33342 (Fig.
metabolically competent rat hepatocytes, enabling 1A & B), Mitotracker (Fig. 1A & C) and histology mechanistic and pathway analyses to be performed. In staining (Fig.1A & D) showed measureable changes.
Fig.4. Multiple homogeneous assay readouts following the exposure addition, correlation between conventional staining, Hoechst showed statistically significant increases in of primary rat hepatocytes to Staurosporine for 4 hours: comparison to morphological changes and sub toxic responses have punctate staining of the nucleus with increases in dose.
Alamar Blue assay. Values are means +/- 1 SD of n=16. been clearly demonstrated using the IN Cell Analyzer.
Mitotracker showed a decrease in the characteristic punctate stained cytoplasm with increasing dose. The Fig. 2. Multiple homogeneous assay readouts following the Mitotracker
P450 reductase histological stain showed a quantitative exposure of primary rat hepatocytes to Staurosporine for 4 hours: increase in the intensity of staining in the cytoplasm comparison to Alamar Blue assay. Values are means +/- 1 SD of Transmitted
Staurosporine and Diclofenac were obtained from light
Cell number
Sigma, UK. Staurosporine was used at final Alamar Blue
concentrations of 0.5, 1 and 5 µM and Diclofenac at Exposure of the hepatocytes stained with Annexin V Results expressed as percentage of control values ± SD. and Hoechst to Staurosporine for 4 hours (Fig. 3) 300, 150 and 50µM. Black, 96 well collagen type IV showed that at all doses there was an increase in Table 1. Five readings following the exposure of primary rat coated plates were obtained from Biocoat, UK. Plates hepatocytes to Diclofenac 25 h using the IN Cell Analyzer 3000: were dosed with Diclofenac prior to seeding the cells punctate staining in the nucleus as well as an increase in cell membrane staining for phosphatidylserine (Fig.
4). Apoptosis can be quantitated in a mechanistic until required. Staurosporine treated cells were seeded manner more sensitively with respect to cell number Nuclear condensation increased significantly at 300 µM, for 21 hours prior to a 4 hour exposure. Primary rat concomitant with decreases in mitochondrial punctate hepatocytes were obtained freshly isolated from and Alamar Blue assay measurements, with no staining and a reduction in NBT reductase staining. No Bowman Research, (UK) Ltd. Cells were accepted if increase in time, but increases in sensitivity andinformation content. Similar results were obtained for changes were detectable with Alamar Blue although cell the viability ≥ 80 % on arrival. Cells were cultured at cells dosed with Diclofenac for 25 hours, following the 30,000 cells per well in a final volume of 200µl growth media, in an humidified incubator at 37 C 5% CO2/95 The assays measured on the IN Cell Analyzer 3000 % air for 25 hours. Primary rat hepatocyte growth provided sensitive measurements of apoptosis, with respect media: 1:1 mix, Williams' E and Hams F12 with to the mitochondria and nucleus, as well as cell number additions of 5 µg/ml insulin, 5 µg/ml transferrin, 5 µg/ml measurements and an indication of metabolic competence.
selenium, 10nM dexamethasone, 2% foetal calf serum, The sensitivity of the measurements appeared to be 10mM HEPES, 50U.ml penicillin G, 50µg/ml increased with respect to the Alamar Blue cytotoxicity streptomycin sulphate. All tissue culture reagents were assay. Four endpoints were measured in each well with no obtained from GIBCO Brl, UK, unless otherwise stated.
increase in time, but a large increase in information content.
Fig.1. Images captured from IN Cell Analyzer 3000. Primary rat Fluorescent probes Hoechst 33342, Mitotracker deep hepatocytes following 4 hour exposure to Staurosporine. A: red (633) and Annexin V (red) were obtained from composite readout using red (Mitotracker deep red) and blue Demonstrates use of IN Cell Analyzer in live Molecular Probes. Draq 5 was obtained from Biostatus (Hoechst 33342) laser lines and transmitted light (histology stain) images. Highlighted area is shown individually as the (B) blue, Ltd. Probe mixtures were made up in warmed (C) red and (D) transmitted light channels respectively. Images hepatocyte growth media, and dosed in a final volume Fig.3. Images captured from IN Cell Analyzer 3000. Primary rat can be analyzed in real time or offline. of 100µl for 15 min. NADPH dependent nitro blue hepatocytes following 4 hour exposure to Staurosporine. A: Control culture stained using Hoechst 33342 (blue) and Annexin tetrazolium reductase was used as descrbed by Cells were found to be viable at all doses (Fig. 2), cell V (red). B: Culture dosed with 1µM Staurosporine, stained using Murphy et al. (Methods in Cell Science, 21:31-38.1999)
number dropping to approximately 80% of control Hoechst 33342 (blue) and Annexin V (red). values at the highest dose. The three measurements IN Cell assays have increased sensitivity whencompared to traditional assays e.g. Alamar Blue.
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