CytoSelect™ 24-Well Cell Migration Assay (5 μm, Fluorometric Format)

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Reactivity
Mammalian
Application
Cellular Assay (CA)
Options
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Supplier Product No.
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Brand CytoSelect™
Sample Type Serum, Cell Samples
Analytical Method Quantitative
Detection Method Fluorometric
Characteristics CytoSelect™ Cell Migration Assay Kit utilizes polycarbonate membrane inserts (5 μm pore size) to assay the migratory properties of cells. The kit does not require you to prelabel the cells with Calcein AM or remove non-migratory cells (i.e. cotton swabbing). Any migratory cells are first dissociated from the membrane, then lysed and detected by the patented CyQuant® GR Dye (Invitrogen). CytoSelect™ Cell Migration Assay Kit provides a robust system for the quantitative determination of cell migration. This Trial Size kit contains sufficient reagents for the evaluation of 4 samples. The 5 μm pore size is optimal for monocyte and macrophage cell migration. However, in the case of epithelial and fibroblast, a larger pore size (8 μm) is recommended. For neutrophil chemotaxis, a smaller pore size (3 μm) is recommended. The CytoSelect™ Cell Migration Assay Kit contains PET membrane inserts (5 μm pore size) in a 24- well plate. The membrane serves as a barrier to discriminate migratory cells from non-migratory cells. Migratory cells are able to extend protrusions towards chemoattractants (via actin cytoskeleton reorganization) and ultimately pass through the pores of the polycarbonate membrane. These migratory cells are then dissociated from the membrane and subsequently detected by the patented CyQuant® GR Dye (Invitrogen). 2
Components
  1. 24-well Migration Plate : One 24-well plate containing 4 cell culture inserts (5 μm pore size)
  2. Cell Detachment Solution : One 10 mL bottle
  3. 4X Lysis Buffer : One 2 mL tube
  4. CyQuant® GR Dye : One 10 μL tube
  5. Forceps : One each
Material not included
  1. Migratory cell lines
  2. Cell culture medium
  3. Serum free medium, such as DMEM containing 0.5 % BSA, 2 mM CaCl2 and 2 mM MgCl2
  4. Cell culture incubator (37 °C, 5 % CO2 atmosphere)
  5. Light microscope
  6. 96-well plate suitable for a fluorescence plate reader
  7. Fluorescence plate reader
Background Cell migration is a highly integrated, multistep process that orchestrates embryonic morphogenesis, tissue repair and regeneration. It plays a pivotal role in the disease progression of cancer, mental retardation, atherosclerosis, and arthritis. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of the attractant, these protrusions can consist of large, broad lamellipodia or spike-like filopodia. In either case, these protrusions are driven by actin polymerization and can be stabilized by extracellular matrix (ECM) adhesion or cell-cell interactions (via transmembrane receptors).
Application Notes Optimal working dilution should be determined by the investigator.
Comment

  • Fully quantify chemotaxis with no manual cell counting
  • Measure chemotaxis in less than 6 hours with most cell types
  • Membrane inserts are uncoated to allow use with any chemoattractant

Assay Procedure
  1. Under sterile conditions, allow the 24-well migration plate to warm up at room temperature for 10 minutes.
  2. Prepare a cell suspension containing 0.5-5.0 x 106 cells/mL in serum free media. Agents that inhibit or stimulate cell migration can be added directly to the cell suspension.
  3. Add 0.5 mL of media containing 10 % fetal bovine serum or desired chemoattractant(s) to the lower well of the migration plate.
  4. Add 100 μL of the cell suspension solution to the inside of each insert.
  5. Incubate for 1-24 hours in a cell culture incubator.
  6. Carefully aspirate the media from the inside of the insert. Transfer the insert to a clean well containing 400 μL of Cell Detachment Solution. Incubate 30 minutes at 37 °C. 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. 4
  7. Completely dislodge the cells from the underside of the membrane by gently tilting the insert several times in the detachment solution. Remove and discard the insert.
  8. Transfer 400 μL of the 0.5 mL medium solution containing migratory cells (step 5) to the well that contains 400 μL of Cell Detachment Solution for the same migration assay sample (step 7). Mix well, transfer 180 μL of the mixture to a 96-well plate. Note: This step combines cells that migrated through the membrane and into the medium, and migratory cells detached from the bottom side of the membrane by Cell Detachment Solution.
  9. Prepare sufficient 4X Lysis Buffer/CyQuant® GR dye solution for all samples by diluting the dye 1:75 in 4X Lysis Buffer (for example, add 5 μL dye to 370 μL of 4X Lysis Buffer).
  10. Add 60 μL of 4X Lysis Buffer/CyQuant® GR dye solution to each well of the 96-well plate containing migratory cells. Incubate 20 minutes at room temperature.
  11. Transfer 200 μL of the mixture 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
Storage 4 °C
Storage Comment Store all components at 4°C.
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