The BioFlux 200 System Using Well Plate Microfluidics for Live Cell Assays Product Overview and Tutorial

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1 The BioFlux 200 System Using Well Plate Microfluidics for Live Cell Assays Product Overview and Tutorial

2 Introduction to the BioFlux System Enables live-cell assays with precisely-controlled shear flow Uses a benchtop instrument to control shear flow and temperature Runs the BioFlux Well Plate Microfluidics consumables Standard well-plate format for convenience Integrated microfluidic flow-channels on the bottom for physiologically-relevant experiments Utilizes BioFlux Software control and analysis Easy protocol control from intuitive graphical interface Programmable image acquisition capabilities Image analysis modules for common data analysis routines

3 Many Applications Require Shear Flow Inflammation/Vascular Biology Platelet adhesion Cellular adhesion strength Leukocyte adhesion Rolling adhesion Transmigration Cancer Biology Cancer/Endothelial cell adhesion and migration Tissue adhesion Metastasis Pharmacology Adhesion characterization Drug binding assays (IC 50 ) Flow-dependent differentiation Stem cell morphology Neuronal growth /morphology Renal cells Microbiology Microbial biofilms Host pathogen interactions Microbial adhesion Parasitology

4 BioFlux Principle Feature Set Applies constant shear to simulate physiological conditions Easily runs up to 24 assays per plate Simple setup without the need for sterilization of any components Simple automation protocols Minimal use of drug compounds and media (20µL - 3mL of media/well ) Can run experiments under different microenvironmental conditions

5 Hardware: Main Components CCD Camera 2. Inverted microscope (can use existing scope or purchase with system) 3. Pressure Interface 4. BioFlux Controller 5. BioFlux Plate 6. Heating Plate

6 BioFlux Core Technology: Well Plate Microfluidics Convenience of a well plate Standard microplate footprint Compatible with liquid handling workstations Suitable for high content analysis High content data of a flow cell Plate bottom replaced with microfluidic channels Liquid travel from wells to channels In a microfluidic format Low reagent use (ul) Controlled shear flow

7 BioFlux Plates 48-well Plate 24 flow channels (one input well each) 24-well Plate 8 flow channels (two input wells each) 180µm glass bottom (standard cover slip) Flow channels: 70µm tall x 350µm wide

8 Operating Principal Cells and reagents are added directly to the wells of the plate. 2. The pressure interface attaches to the plate by tightening the 4 clamps. 3. As pressure is applied from the instrument, it moves the cells/reagents through the bottom of the well and into the fluidic channels. The fluid will accumulate in the waste well.

9 How to Acquire Images Place the BioFlux plate (with interface on top) on a standard inverted microscope. Compatible with microplate holders, manual stages, and automated X-Y stages Focus on one of the viewing windows to begin image acquisition. 3. The sample to be imaged sits right on top of the 180um cover glass, which forms the bottom of the microplate. The thin cover glass bottom makes the plates compatible with brightfield, phase, DIC, epifluorescence, and confocal microscopy.

10 In the Viewing Window BioFlux shear stress profile bottom surface Uniform, laminar flow within the region of interest

11 Typical Cell Adhesion Workflow: Ligand Based 1. Introduce coating into channels (10 min) (incubation period 1 hour) 2. Wash with saline buffer (5 min) 3. Block plate with BSA or add antibody (10 min) 4. Add cells (10 min) 5. Wash cells- (10 min) 6. Image- (10 min) Compatible with manual or automated liquid handling

12 Typical Cellular Adhesion Workflow: Cellular Monolayer Option Important foundation for: cell culture, cell adhesion, rolling, transmigration, morphology studies full width of channel CHO, 45 min after seeding HUVEC grown to 100% confluence Platform supports a wide variety of biological matrices for cell growth

13 BioFlux Software Manual Mode Supports assay development and experimentation with varying shear forces, times, etc. Set shear values directly in the control panel Click on wells to establish flow

14 BioFlux Software AutoRun Mode Easily creates and runs protocols which automate your commonly used assays Assemble flow protocols into run tables to create a customized sequence of events. Create and edit flow protocols with varying time, shear flow, and compound exchange. Also controls image acquisition settings.

15 BioFlux Software Analysis Mode Analyzes images for intensity, cell counting, and velocity in a simple, intuitive manner. Data compilation is recorded and exportable for archiving. Modules available for cell counting, fluorescence intensity, particle tracking, and more.

16 Representative Data Cell Adhesion Adhesion data under flow conditions Top channel shows cells adhering to coated channel. Bottom channel shows block of adhesion with antibody Dose-response curves produced for three different antibodies

17 Representative Data Cell Adhesion Rolling velocity calculations Image Stacker module compiles images into stacked frames Select a track to automatically calculate distance and rolling velocity Contour Distance (um) Velocity (um/s) AVERAGE STD

18 Representative Data Biofilms Biofilms 24 simultaneous biofilm experiments run under identical conditions, added different anti-microbials Live/dead staining inside the channels and subsequent ratiometric analysis Extracted effluent and determined CFU s by traditional microbiology techniques

19 Fluxion Biosciences For more information, please contact: Fluxion Biosciences (866) Toll Free 384 Oyster Point Blvd. #6 South San Francisco, CA 94080