CytoSelect™ Leukocyte Transmigration Assay - Product Manual - 24 assays

Aug 26, 2026

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CytoSelect™ Leukocyte Transmigration Assay is a research-use kit for quantifying leukocyte–endothelium interactions and transmigration as a multi-step inflammatory recruitment process. The protocol uses TNFα to activate endothelial monolayers, tracks leukocytes with LeukoTracker™, and measures migrated cells through a 3 µm pore insert in a 24-well format. Fluorescence readout at 480/520 nm supports comparative assessment across conditions, with reagents sufficient for 24 assays.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages7

Reading Time18 min

AccessFree to download

Introduction

Assay Principle

Related Products

Kit Components and Storage

Materials Not Supplied

Storage

Assay Protocol

Example of Results

References

Product Manual CytoSelect™ Leukocyte Transmigration Assay Catalog Number CBA-212 24 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Leukocyte extravasation into perivascular tissue plays a key role in inflammatory diseases. This recruitment requires leukocyte interaction with vascular endothelium and consists of multiple, consecutive processes including the capture of circulating leukocytes, subsequent leukocyte rolling, arrest, firm adhesion and transmigration (Figure 1) . This multistep paradigm is realized by sequential activation-dependent interactions between endothelial cell adhesion molecules and their specific ligands on leukocytes. The first step of transient adhesion and rolling is known tobe mediated by an interaction of leukocyte or endothelial cell selectins and their oligosaccharide-bearing ligands. Arrest and firm adhesion of leukocytes to endothelium is dependent on the activation of β2 integrins like Mac-1 or LFA-1 on the leukocyte cell surface, followed by interaction with endothelial cell proteins belonging to the Ig superfamily such as ICAM-1. Cell Biolabs ’ CytoSelect™ Leukocyte Transmigration Assay provides a robust system for the quantitative determination of leukocyte-endothelium interactions and transmigrations. The kit contains sufficient reagents for the evaluation of 24 assays in a 24-well plate format. Figure 1. The Leukocyte Adhesion Cascade Assay Principle Related Products 1. CBA-100: CytoSelect™ 24-Well Cell Migration Assay (8μm, Colorimetric) 2. CBA-101: CytoSelect™ 24-Well Cell Migration Assay (8μm, Fluorometric) 3. CBA-210: CytoSelect™ Leukocyte-Endothelium Adhesion Assay 4. CBA-216: CytoSelect™ Tumor Transendothelial Migration Assay Kit Components (shipped at room temperature) 1. 24-well Migration Plate (Part No. 121203): Two 24-well plates, each containing 12 cell culture inserts (3 µm pore size) 2. 500X LeukoTracker™ Solution (Part No. 12101): One 100 µL tube 3. 4X Lysis Buffer (Part No. 10404): One 10 mL bottle 4. TNFα (Part No. 12105): One 100 µL tube of 10 µg/mL TNFα in sterile 1X PBS/0.1%BSA 5. Cotton Swabs (Part No. 11004): 40 each 6. Forceps (Part No. 11005): One each Materials Not Supplied 1. Endothelial cells and cell culture medium 2. 24-well tissue culture plate 3. Serum free medium, such as RPMI containing 0.5% BSA, 2 mM CaCl2 and 2 mM MgCl2 4. Cell culture incubator (37ºC, 5% CO2 atmosphere) 5. 1X PBS containing 2 mM CaCl2 and 2 mM MgCl2 6. Light microscope 7. 96-well plate suitable for a fluorescence plate reader 8. Fluorescence plate reader Storage LeukoTracker™ Solution and TNFα should be removed from the kit and stored at-20ºC immediately. Store all other components at 4ºC. Assay Protocol 1. Add 50,000-100,000 endothelial cells in 100 µL medium to each insert in a 24-well plate containing 500 µL of culture medium. 2. Culture cells for 48-72 until the endothelial cells form a monolayer. 3. Treat endothelial cell monolayer with desired activator or inhibitor, such as TNFα . 4. Harvest leukocytes and prepare a cell suspension at 0.5-1.0 x 106 cells/ml in serum free media. 5. Add LeukoTracker™ to a final concentration of 1X (for example, add 2 µL of 500XLeukoTracker™ solution to 1.0 mL of leukocyte cell suspension) . Incubate for 60 min at 37ºC in a cell culture incubator. Spin down cells at 1000 rpm for 2 minutes, aspirate the medium and wash cell pellet with serum free media. Repeat the wash twice. Resuspend the cell pellet at 0.25-1.0 x 106 cells/ml in serum free media. Agents that inhibit or stimulate cell migration may be added directly to the cell suspension. 6. Carefully remove endothelial culture medium from migration insert without disturbing the endothelial monolayer and transfer the insert to another well containing 500 µL of leukocyte culture media including desired chemoattractant(s) . 7. Add 100 µL ofthe cell suspension solution to the inside of each in

FAQs

What biological process does the assay measure?

It quantifies leukocyte-endothelium interactions and transmigration, reflecting consecutive recruitment steps such as rolling, arrest/adhesion, and movement through the endothelial barrier.

How are leukocytes detected in the assay?

Leukocytes are labeled with LeukoTracker™ at a final 1X concentration, incubated at 37°C, then washed and allowed to migrate toward chemoattractants.

How is the migration read out and quantified?

After removing non-migratory cells, migrated cells are lysed in 4X Lysis Buffer. The resulting lysate is transferred to a fluorescence plate and measured with a fluorescence plate reader at 480 nm/520 nm.

Aug 26, 2026

Upload by Ophelia