Stencil devices create the exclusion zone by physical occlusion during cell seeding, rather than mechanical disruption after confluence. This avoids substrate damage and gives you a consistent, defined gap width across every replicate. It removes a major source of well-to-well variability found in manual scratch wound assays.
Cell-based Analysis
Cell Imaging Dyes, Stains, and Fluorescent Organelle Probes
Cell imaging dyes, stains, and confinement devices for precise live-cell fluorescence imaging
You have two toolkits for live-cell imaging: probes that reveal structure, and devices that hold your cells still. Reach for fluorescent organelle probes when you want to label live cells without fixation. Choose microstructured devices when you need to control cell position, adhesion geometry or mechanical confinement during imaging.
Choose PDMS (silicone) stencils to define discrete culture areas on coverslips and multi-well plates, 250 um thick. Pick a single ring-shaped well, or take a multi-area format with 2, 4 or 9 isolated zones on one coverslip. The stencil blocks cell adhesion outside the zone you define. Peel it away before fixation to leave a clean field for imaging. Use this for wound-healing assays and co-culture work, where you want precise spatial boundaries without manual scratching. Pick your format: Solo (single well), Quartet (4-well), Nonet (9-well) or Presto/Allegro (2 oblong wells).
Cell-permeant fluorescent probes label organelles in live cells. No genetic reporters or fixation steps required. Choose an endoplasmic reticulum probe when you want clear ER morphology under standard fluorescence filters. The rhodamine-based (TMR) ER probe is red-emitting and reads on standard TRITC or DsRed filter sets. Add it directly to your culture medium at around 50 uM for stable ER fluorescence over about 3 hours. Image it in your live cells: the ER signal cannot survive fixation.
Use agarose-based compression devices to apply controlled, uniform confinement and study how your cells respond over short and long timeframes. Silicon wafers mould the agarose pillars to a set height. Agarose spans roughly 1 to 150 kPa, close to the in vivo range. You get confinement for up to 10 days. A uniform cell height also steadies your imaging in thick samples or cells with uneven attachment. Pick your pillar height from about 0.5 to 200 um in 0.5 um steps, to match the confinement you want against your cell type.
Choosing the Right Cell Imaging Tool
- You need to run a wound-healing or gap-closure assay with a defined exclusion zone. Use PDMS (silicone) stencil culture devices. Apply the stencil before seeding, grow your cells to confluence around it, then peel the stencil to create a reproducible gap. No scratch is needed, and the exclusion zone stays geometrically defined and consistent across replicates.
- You are imaging co-cultures or multi-condition experiments in a single well. Choose a multi-area stencil format for your 4 or 9 isolated zones on one coverslip. Each area stays separate during culture and appears in the same field once you remove the stencil.
- You need live-cell endoplasmic reticulum labelling without genetic modification. Use a cell-permeant ER fluorescent probe. It labels your ER network specifically. The referenced red-emitting, rhodamine-based probe reads on standard TRITC or DsRed filter sets in live cells, with no fixation needed.
- You want to study cell behaviour under mechanical confinement, or your cells are hard to image in a single focal plane because of irregular attachment or thickness variation. Use an agarose-based confinement device, moulded with silicon wafers, to apply defined, uniform confinement. Select the confinement height to match the degree of confinement you want against your cell type, for a more consistent z-plane across your field.
Applications
Wound healing assays
PDMS (silicone) stencils create a defined exclusion zone for scratch-free gap closure. Pair with cell tracking reagents to follow movement into your cleared zone.
Co-culture experiments
Multi-zone stencils hold two, four or nine cell populations on one coverslip. Compare paracrine signalling and cell behaviour in your cultures side by side.
Live-cell organelle imaging
Cell-permeant ER probes label your endoplasmic reticulum without fixation. The probe reads on standard TRITC or DsRed filters on most digital imaging systems.
High-content imaging standardisation
Agarose-based confinement devices hold your cells to a defined height, cutting focal variability across the plate for consistent, comparable measurements.
Post-fixation immunofluorescence
Stencil geometry survives standard paraformaldehyde fixation, so your gap stays visible alongside normal immunostaining in the same fixed sample.
Multicolour organelle imaging
Pair red-emitting ER probes (TRITC/DsRed) with spectrally distinct probes (e.g. far-red mitochondrial dyes) for your multi-organelle imaging and flow cytometry work.
Frequently asked questions
Standard paraformaldehyde fixation after stencil removal works with most PDMS stencil systems. Fix and immunostain your cells in the normal way. The defined gap geometry stays intact, so you can measure gap closure alongside marker staining in the same sample. Live ER-probe signal is the exception: it is not fixable and must be imaged before fixation.
Yes, as long as the ER probe's excitation and emission spectrum is compatible with your co-stains. The referenced red-emitting, rhodamine-based ER probe is detected with standard TRITC or DsRed filters. Pair it with spectrally distinct mitochondrial probes or nuclear dyes for multi-organelle imaging on standard multicolour filter sets.
Confinement heights span a wide range. For the agarose-based system, wafer and pillar sizes run from about 0.5 to 200 um in 0.5 um increments. Select the height to match your cell type and the degree of confinement you want, for the most consistent, reproducible result.
Parent category: Cell-based assay kits
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