Simplify Cell Analysis With Integrated Imaging and Plate Reading Technology

Get a front row view of your cells See more, and do more, with an imaging cytometer that enables rapid imaging and cell analysis, giving you a better view of cell proliferation, marker expression, and cytotoxicity. Count and measure the confluency of your cells without labeling them using StainFree technology. Discover how you can simplify your workflow by combining plate reading and imaging in a small footprint. For more information, visit www.moleculardevices.com Wells to Westerns: Investigating the cellular heat shock response ...............................2 StainFree technology for cellular image analysis.......................................................... 3 Alternatives to DAPI staining: imaging and counting live cells ........................................... 4 Improved cell counting method using transmitted light............................................5 Measuring marker expression with imaging cytometry on a plate reader.................6 Cell migration analysis with Oris Pro assay...... 7 Assay apoptosis in magnetically bioprinted HepG2 spheroids................................ 8 Normallize HTRF cytokine assays to cell viability...........................................................9 eBook contents www.moleculardevices.com | 2 • Monitor cell proliferation without dyes • Investigate mechanisms of cell death with fluorescent cell imaging • Image cells, run cell-based assays, and detect western blots, all on one platform Wells to Westerns: Investigating the cellular heat shock response Investigating a cellular response can involve multiple approaches to gathering information—imaging, cell-based viability and proliferation assays, western blots to look at changes in protein expression, and more. Often multiple instrument platforms are needed to glean the necessary results, and several software packages may need to be learned in the process. READ APPLICATION NOTE  StainFree measurement of cell proliferation after heat shock. On the left are a transmitted light image of untreated CHO-K1 cells (top) and purple masks showing cells identified by StainFree analysis (bottom). Graphs show StainFree cell counts for normal and stressed populations of cells under control or heat shocked conditions. Cell counts were compared at 6 and 24 hours. Normal StainFree cell counts StainFree cell counts Stressed Normal Stressed 6 hours Stressed 24 hours Control Heat shocked Control Heat shocked www.moleculardevices.com | 3 StainFree technology for cellular image analysis without fluorescent dyes Imaging cell-based assays typically requires the use of fluorescent probes that can be toxic to living cells or may only function in fixed cells. A label-free method for analyzing cell counts and cell confluence enables researchers to quantitatively monitor cell proliferation and health without time-consuming workflows that may disrupt cell viability. READ APPLICATION NOTE  • Count cells and estimate confluence without fluorescent dyes • Monitor cell growth without disrupting cells • Set up imaging assays quickly and easily with the intuitive SoftMax® Pro Software user interface StainFree technology compared to fluorescence cell counting. Cells counted using StainFree technology (blue), red nuclear stain (red), and green whole-cell stain (green). Counts obtained from all three methods agree closely, demonstrating that StainFree technology gives accurate cell counts while eliminating the need for fluorescent dyes (R2 > 0.99 for each plot). IC50 curves for cytotoxic compounds. Cell counts were performed using StainFree technology and plotted vs. compound concentration to obtain IC50 curves. Cells seeded per well Cell count Staurosporine Doxorubicin Mitomycin C Paclitaxel [Compound] (µM) Cell count www.moleculardevices.com | 4 Alternatives to DAPI staining: imaging and counting live cells DAPI (4’, 6-diamidino-2-phenylindole) is a fluorescent dye often used to stain nuclear DNA. It is employed in imaging experiments such as fluorescent microscopy, chromosome spreads, FACS, and cell-based assays. However, excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI fluorescence in the FITC/GFP channel of an imaging system, causing errors in interpretation of results. READ APPLICATION NOTE  • Enable live cell counting while maximizing cell viability with StainFree Technology • Eliminate fixation steps required prior to staining • Save time and money by avoiding costly staining procedures Workflow for cell analysis with StainFree technology vs. Live Red Dye vs. DAPI. The StainFree workflow saves about 70 minutes compared to fixation and staining with DAPI. Moreover, cells analyzed using StainFree technology remain fully viable and can be used in additional assays. Cells counted with the SpectraMax MiniMax 300 Imaging Cytometer. Cells stained with Live Red Dye were imaged in the red fluorescent (top left) or transmitted light (top middle) channel. StainFree cell counts (top right, purple masks) correlate very closely to cell counts based on red nuclear staining, as shown in the graph (green circles, StainFree counts; red circles, red nuclear counts). The r2 values for both curves were 0.99. Fix (30 min) DAPI Wash (5 min) Stain (30 min) Wash (5 min) Image Image StainFree Stain (30 min) Live Red Dye Image Initial seeding density (cells/well) Cell counts www.moleculardevices.com | 5 Improved cell counting method using transmitted light Hematoxylin, a natural product extracted from the heartwood of the logwood tree (Haematoxylin campechianum), is the most commonly used dye for staining protocols in histology. Hematoxylin reacts like a basic dye with a purplish-blue color and stains acidic or basophilic structures, including the cell nucleus and organelles that contain RNA, such as ribosomes and the rough endoplasmic reticulum. READ APPLICATION NOTE  • Improved accuracy of cell counting for difficult-to-image cell types • Economical alternative to fluorescent cell staining • Simple workflow from data collection to analysis Images of unstained MDCK cells in transmitted light (focus at 10 µm). A: The MDCK parental cell line is polymorphic. B: MDCK I cells have nearly indistinguishable cell membranes. C: MDCK II cells are polygonal with clearly defined cell borders and a mosaic-like appearance. Images of parental MDCK cells from the TL channel of the MiniMax cytometer. A: Unstained parental MDCK cells; B: Hematoxylin stained parental MDCK cells; C: Same field as B, with cells identified by the software shown as purple masks (overlays). www.moleculardevices.com | 6 • Expanded plate reader applications with imaging cytometer functionality • Easily measure marker expression levels per cell or per well • Follow a familiar microplate reader workflow • Confidence in the data output with cell visualization Measuring marker expression with imaging cytometry on a plate reader The presence or absence of cellular proteins can indicate a reaction to stimulation, a state of differentiation, unique characteristics of a specific cell type, or success of a gene transfection. For example, the surface expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) is usually absent from normal vascular endothelium but can be stimulated in vitro by the cytokine TNF-α to elicit an inflammatory response. Such protein expressions in a cell can be readily visualized and easily quantified using imaging cytometry. READ APPLICATION NOTE  Cellular response to treatment with anti-inflammatory compounds. Images on the left show cells with fluorescent VCAM-1 expression, plus purple masks on the right show segmentation of cells exhibiting the inflammation marker. Cells treated with a high dose of the anti-inflammatory compound SB202 (top row) show little VCAM-1 expression while cells treated with a very low dose of SB202 (bottom row) were stimulated by the cytokines and expressed a large quantity of VCAM-1. Values are given in normalized total signal intensity. 1.3 27.0 www.moleculardevices.com | 7 Cell migration analysis with Oris Pro assay Cell migration, the movement of cells from one location to another, is a critical component of both normal and abnormal biological processes. The importance of cell migration in diverse biological processes has led to the development of systems for studying the mechanisms responsible in an effort to identify therapeutic components capable of either promoting (wound healing) or inhibiting (tumor formation) cell migration. READ APPLICATION NOTE  Schematic of Oris Pro Cell Migration Assay. This assay uses a non-toxic biocompatible gel to form a cell-free zone in the center of the well. After cells are seeded, the biocompatible gel dissolves, allowing cells to migrate into the detection zone in the center of the well. For images acquired with the MiniMax cytometer, a region of interest corresponding to the detection zone is specified, and the level of cell migration (area covered by cells) is analyzed. • Accurately measure cell migration from a single image per well • Get the flexibility of StainFree or fluorescence image analysis with a large signal window • Automatically analyze region of interest containing migrated cells with SoftMax Pro Software Seed cells around Oris Pro biocompatible gel and allow them to adhere. The biocompatible gel dissolves to create a detection zone. Allow cells to migrate into the detection zone. Measure cells in detection zone via microscopy, imaging instrument, or microplate reader. www.moleculardevices.com | 8 Assay apoptosis in magnetically bioprinted HepG2 spheroids Two-dimensional (2D) cell culture has been used for over a century to investigate a wide range of cellular processes. However, more recent studies have shown that responses in 2D systems can differ significantly from those seen in vivo (Duval et al., 2017). A variety of methods for transforming 2D cultures into 3D spheroids have been devised, but many of these require a long time to form spheroids that can be difficult to handle. READ APPLICATION NOTE  • Magnetically bioprint spheroids in as little as 15 minutes • Center spheroids in flat-bottom microplate wells for rapid 1-site imaging of results • Assess apoptosis in spheroids in less than one hour with a homogeneous, fluorescent assay Overlaid TL and fluorescence images. Spheroids were treated with high (top row) or low (bottom row) concentrations of anisomycin or doxorubicin and assayed with the EarlyTox™ Caspase-3/7-D NucView 488 Assay Kit. Images taken in the TL and green fluorescent channels were overlaid. www.moleculardevices.com | 9 Normalize HTRF cytokine assays to cell viability Pro- and anti-inflammatory cytokines play a central role in autoimmunity and inflammatory and infectious diseases. They are also key players in metabolic disorders and oncology, particularly the anti-tumor immune response. Homogeneous Time Resolved Fluorescence (HTRF) from Cisbio Bioassays provides a versatile platform for the quantitation of various cytokines and chemokines. HTRF is perfectly suited to monitoring cytokine and chemokine release in cell-based experiments, especially physiological models such as peripheral blood mononuclear cells (PBMC). READ APPLICATION NOTE  • Evaluate cytokine secretion accurately with sensitive HTRF technology • Monitor cell seeding on the MiniMax cytometer without staining • Confirm assay quality by monitoring viability of assayed cells with the EarlyTox™ Cell Integrity Assay *To view the complete workflow of how to normalize HTRF cytokine assays to cell viability, please visit moleculardevices.com/htrf-workflow Cytokine secretion upon LPS stimulation, measured by HTRF assays on the SpectraMax i3x Multi-Mode Microplate Reader. Unstimulated LPS-Stimulated [Cytokines] pg/mL hIL1β hIL6 hIL8 hTNFα The trademarks used herein are the property of Molecular Devices, LLC or their respective owners. Specifications subject to change without notice. Patents: www.moleculardevices.com/productpatents FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. ©2020 Molecular Devices, LLC 8/20 2345A Phone: +1.800.635.5577 Web: www.moleculardevices.com Email: [email protected] Check our website for a current listing of worldwide distributors. *Austria, Belgium, Denmark, Finland, France, Germany, Ireland, Netherlands, Spain, Sweden and Switzerland Contact Us Regional Offices USA and Canada +1.800.635.5577 United Kingdom +44.118.944.8000 Europe* 00800.665.32860 Japan +81.3.6362.9109 South Korea +82.2.3471.9531 China (Beijing) +86.10.6410.8669 China (Shanghai) +86.21.3372.1088 Hong Kong +852.3971.3530 SpectraMax® i3x Multi-Mode Microplate Reader with the field-upgradeable SpectraMax® MiniMax™ 300 Imaging Cytometer