CytoSelect™ Leukocyte Transmigration Assay

Catalog No. ABIN2344904

Product Details

Detection Method: Fluorometric

Application: Biochemical Assay (BCA)

Brand: CytoSelect™

Sample Type: Cell Samples, Serum

Analytical Method: Quantitative

Characteristics: 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. Figure 1. The Leukocyte Adhesion Cascade

Components:

1. 24-well Migration Plate : One 24-well plate containing 24 cell culture inserts (3 μm pore size)
2. 500X LeukoTracker™ Solution : One 100 μL tube 3
3. 4X Lysis Buffer : One 10 mL bottle
4. TNFα : One 100 μL tube of 10 μg/mL TNFα in sterile 1X PBS/0.1%BSA
5. Cotton Swabs : 40 each
6. Forceps : One each

Material not included:

1. Endothelial cells and cell culture medium
2. 24-well plate
3. Serum free medium, such as RPMI containing 0.5 % BSA, 2 mM CaCl2 and 2 mM MgCl2
4. Cell culture incubator (37 °C, 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

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Quantify interactions between endothelium and leukocytes
• Fully quantify cell transmigration with no manual cell counting
• Highly sensitive results on a fluorescence plate reader

Assay Procedure:

1. Add 50,000-100,000 endothelial cells in 300 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 or leukocyte cells 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. Agents that inhibit or stimulate cell migration can be added directly to the cell suspension.
5. Add LeukoTracker™ to a final concentration of 1X (for example, add 2 μL of 500X LeukoTracker™ solution to 1.0 mL of leukocyte cell suspension). Incubate for 60 min at 37 °C 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.
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 300 μL of the cell suspension solution to the inside of each insert. 4
8. Incubate for 2-24 hours in a cell culture incubator.
9. Carefully aspirate the media from the inside of the insert. Use cotton-tipped swabs to gently remove non-migratory cells from the interior of the inserts. Take care not to puncture the polycarbonate membrane. Be sure to remove cells on the inside perimeter. Note: Retain the medium in the 24-well migration plate that contains chemoattractant(s) and cells that migrated through the membrane and into the medium.
10. Transfer 400 μL of the 500 μL bottom medium solution containing migratory cells (step 9) to a clean well that contains 150 μL of 4X Lysis Buffer and place the swabbed insert into the same well. Incubate 5 minutes at room temperature with shaking. Note: This step combines cells that migrated through the membrane and into the medium, and migratory cells still attached to the bottom side of the membrane.
11. Transfer 150 μL of the mixture to a 96-well plate suitable for fluorescence measurement. Read fluorescence with a fluorescence plate reader at 480 nm/520 nm.

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: LeukoTracker™ Solution and TNFα should be removed from the kit and stored at -20°C immediately. Store all other components at 4°C.

References

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White, Krishnamoorthy, Gupta, Lancelot, Moore, Sarnaik, Hobbs, Light, Hines: "VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow." in: British journal of haematology, Vol. 174, Issue 6, pp. 970-82, (2016) (PubMed).

Giunzioni, Bonomo, Bishop, Castiglioni, Corsini, Bellosta: "Cigarette smoke condensate affects monocyte interaction with endothelium." in: Atherosclerosis, Vol. 234, Issue 2, pp. 383-90, (2014) (PubMed).

Roe, Orillo, Verma: "West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model." in: PLoS ONE, Vol. 9, Issue 7, pp. e102598, (2014) (PubMed).

Roberts, Robinson: "Mycobacterium tuberculosis infection of human dendritic cells decreases integrin expression, adhesion and migration to chemokines." in: Immunology, Vol. 141, Issue 1, pp. 39-51, (2013) (PubMed).

Zhao, Lee, Zukas, Middleton, Kinder, Acharya, Hall, Rader, Puré: "CD44 expressed on both bone marrow-derived and non-bone marrow-derived cells promotes atherogenesis in ApoE-deficient mice." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 28, Issue 7, pp. 1283-9, (2008) (PubMed).

Fava, Alpini, Rychlicki, Saccomanno, DeMorrow, Trozzi, Candelaresi, Venter, Di Sario, Marzioni, Bearzi, Glaser, Alvaro, Marucci, Francis, Svegliati-Baroni, Benedetti: "Leptin enhances cholangiocarcinoma cell growth." in: Cancer research, Vol. 68, Issue 16, pp. 6752-61, (2008) (PubMed).

Target Details

Background: 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 to be 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.