Cultrex® Rat Collagen 1 (Trevigen 3440-100-01) is supplied in Acetic Acid (20 mM) at a pH of 3.4 - 3.6. To induce polymerization the collagen solution must be neutralized by mixing with a neutralizing buffer. We recommend the following neutralizing buffer; DMEM (Sigma D2429) + 7.5 g/L sodium bicarbonate + 0.004 g/L folic acid + 1% GlutaMax). When preparing collagen 1 it’s important that it is properly neutralized to ensure cell health is maintained and that the gelling is uniform. Neutralization takes time and requires careful and complete mixing. Neutralize once in a larger volume and then dispense in to the ClearView insert wells - this will help to minimize wastage from dead volume. Once neutralized, Collagen 1 will slowly gel even when kept cold (i.e. within 30 minutes). Working quickly and efficiently will help ensure good results.
The required biomatrix concentration will be dependent on the matrix and cell types used. For HT-1080 cells we recommend Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract (5 mg/mL) and Cultrex® Rat Collagen 1 (1 mg/mL). If you need to optimize the gel density, we recommend testing a range of concentrations that bracket the suggested densities above (e.g., a range of 7.5 to 2.5 mg/mL BME). Over dilution of the biomatrix may impair its ability to polymerize and form an effective 3D gel resulting in migration rather than cell invasion being monitored.
As biomatrix materials are temperature sensitive we recommend using a cooled centrifuge (set at 4°C) to prevent premature, or non-uniform, polymerization. However, many users have successfully performed the chemotactic invasion assay using a centrifuge at ambient temperature. If doing this we would recommend that you work efficiently to minimize the time the plate is at ambient temperature.
We have compared the addition of modulatory compounds within the biomatrix:cell layer and assay medium overlay versus addition to the assay medium overlay alone, and found no significant difference in response between the assay conditions. Although addition of the test agent to the biomatrix:cell layer is more technically challenging you may want to investigate this if you feel your treatments have distinct physical properties which could impair permeation through the biomatrix gel.
We recommend using a CoolBox system (Cat. No. 4444) which provides a consistent temperature of 4-8°C for hours. CoolBox units are insulated boxes that hold a cold pack and either a microplate insert (CoolSink) or micro centrifuge rack (CoolRack), ensuring that the temperature across the ClearView Chemotaxis plate is evenly maintained, thus reducing edge effects, and preventing matrix materials from polymerizing prematurely. Alternatively, crushed ice can be used however non-uniform cooling can lead to assay variability.
We recommend placing the ClearView plate on a pre-warmed CoolSink and incubating at 37°C for 30-60 minutes. The warm CoolSink provides contact with the underside of the ClearView plate and ensures uniform heating and polymerization. Incubating for less than 30 minutes may not allow for complete polymerization. Incubating for longer than 60 minutes may dry the biomatrix gel and result in poor assay performance
We recommend a biomatrix volume of 20 µL per insert well and have optimized the chemotaxis cell invasion protocol for use with this volume. Altering this volume may hinder uniform cell distribution within the biomatrix, cause uneven biomatrix coverage across the membrane or impair imaging acquisition.
Both phase contrast (label-free) and fluorescent cell images can be captured and analyzed during the chemotaxis invasion assay. As cells invade through a 3D biomatrix they tend to elongate and send out filapodia-like projections to remodel and penetrate the matrix. This distinct morphology is associated with thin, high contrast cell bodies within a partially textured gel. In contrast, cells that have reached the bottom surface of the membrane tend to flatten out and have lower contrast. This range of morphologies can confound analysis of bottom-side phase contrast images. As such, we recommend using nuclear labeled fluorescent cells in conjunction with fluorescence analysis for highly accurate and reliable results.
Yes. Cells can be labelled with either a green or red nuclear restricted fluorophore.
The IncuCyte™ Scratch Wound assays measure the movement of cells into a cell-free zone in the absence of a chemotactic gradient. You can use the WoundMaker™ to create a precise wound in each well and monitor wound closure as cells move either across a substrate (migration) or through a 3D gel matrix (invasion). The IncuCyte™ Chemotaxis assay measures directed cell migration in response to a chemotactic gradient. You can use the IncuCyte™ Chemotaxis Cell Migration system to investigate treatment effects on chemotactic profile. The system is suitable for adherent and non-adherent cells.
No. The ClearView cell migration insert has been specifically designed to work with the ClearView reservoir plate.
No. The IncuCyte™ FLR instrument does not have the ability to acquire or process the images fast enough
Yes. The ClearView Cell Migration plate has been extensively validated with non-adherent cell types. To ensure uniform cell settling within each well after seeding. We recomend allowing the plate to sit at room temperature for 45-60 minutes after plating. The Chemotaxis (Top Only) “Analysis Job Type” is recommended for quantifying non-adherent cell migration.
Key variables to optimize include membrane coatings, assay medium and cell density. Recommended experimental conditions for a range of cell types, including cancer cells, vascular cells, and leukocytes, can be found in the assay protocol.
Yes. Both the top and the bottom membrane surfaces can be coated independently or together. Recommended volumes are 20 μL for the top side of the membrane and 150 μL for the bottom side of the membrane.
Yes. We recommend using a nuclear restricted fluorophore (as opposed to cytoplasmic) to ensure neighboring cells can be differentiated and counted as individual objects following image analysis. Fluorophore excitation and emission spectra must be compatible with IncuCyte ZOOM® fluorescent channels.
Yes. By labelling different cell types with fluorescent probes and using the phase, green and red data acquisition channels of IncuCyte ZOOM® it is possible to quantify chemotaxis of more than one cell type in co-culture.
Yes. The IncuCyte ZOOM® Live-Cell Imaging platform does not disturb your cells during the assay. Once the experiment is completed, simply remove the medium from the wells to perform additional analyses.
Yes. Multiplexed measurements of cell health can be made using any unused imaging channel. The IncuCyte ZOOM® instrument supports HD phase-contrast, green fluorescence and red fluorescence automated imaging modes.
No. The optical quality of the membrane surfaces in existing Boyden chambers is not amenable to IncuCyte® imaging or image processing algorithms.
No. Our fluorescence analysis algorithms have been specifically designed to identify nuclear fluorescence in the chemotaxis assay.