CytoSelect™ 24-Well Cell Migration Assay, 3 µm

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Suspend 1.5 × 106 neutrophils or 0.4 × 106 keratocytes in serum-free medium	Incubate for 18 hours in a cell culture incubator.

Upstream tips
Suspend 1.5 × 106 neutrophils or 0.4 × 106 keratocytes in serum-free medium
Protocol tips
Incubate for 18 hours in a cell culture incubator.

Publication protocol

CytoSelect 24-Well Cell Migration Assay (Cell Biolabs, San Diego, CA) (transwell assay) was used to assess SP-induced chemotaxis, involvement of PI3K in SP-enhanced migration, and the role of IL-8 in keratocyte migration. Isolated human neutrophils and keratocytes were used as migratory cell populations. Two different membrane pore sizes were used: 3 µm for neutrophils and 8 µm for keratocytes. The assay was used according to the manufacturer’s protocol. Briefly: 500 µl of supernatants collected from cell treatments or fresh medium supplemented with SP or different concentrations of IL-8 was added to the lower well of the migration plate. Suspension of either 1.5 × 106 neutrophils or 0.4 × 106 keratocytes in serum-free medium was added to the inside of each insert. Plates were incubated for 18 hours in a cell culture incubator. After incubation time, media from the inside of the insert were pirated and the insert transferred to a clean well containing Cell Detachment Solution (Cell Biolabs) and incubated for 30 minutes at 37°C. Cells were dislodged from the underside of the membrane by tilting the insert several times in the detachment solution. Medium from the lower well of the plate was transferred to the well containing Cell Detachment Solution for the same migration assay sample (only neutrophils assay); 180 µl of the mixture was transferred to a 96-well plate and treated with lysis buffer and CyQuant GR dye solution for 20 minutes at room temperature. Mixture was transferred into 96-well plate suitable for fluorescence measurement (Corning) and fluorescence was read with a Synergy HT plate reader at 480/520nm (BioTek, Winooski, VT).

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Download the product protocol from Cell Biolabs for CytoSelect™ 24-Well Cell Migration Assay, 3 µm below.

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