CytoSelect™ 24-well Cell Invasion Assay, Colorimetric

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Säugetier
Applikation
Cellular Assay (CA)
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Marke CytoSelect™
Proben Serum, Cell Samples
Analytische Methode Quantitative
Nachweismethode Colorimetric
Produktmerkmale CytoSelect™ Cell Invasion Assay Kit utilizes basement membrane-coated inserts to assay the invasive properties of tumor cells. It contains sufficient reagents for the evaluation of 12 samples.
Bestandteile
  1. ECM Invasion Chamber Plate : One 24-well plate containing 12 ECM-coated cell culture inserts.
  2. Cell Stain Solution : One 10 mL bottle
  3. Extraction Solution : One 10 mL bottle
  4. Cotton Swabs: 40 each
  5. Forceps: One each
Benötigtes Material
  1. Invasive 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 microtiter plate
  7. Microtiter plate reader
Hintergrund The ability of malignant tumor cells to invade normal surrounding tissue contributes in large part to the significant morbidity and mortality of cancers. Invasiveness requires several distinct cellular functions including adhesion, motility, detachment, and extracellular matrix proteolysis. Metastatic cells produce many proteolytic enzymes (e.g. lysosomal hydrolysates, collagenases, plasminogen activators) while the expression of certain cell surface protease receptors is also increased.
Applikations-hinweise Optimal working dilution should be determined by the investigator.
Kommentare

  • Fully quantify cell invasion with no manual cell counting
  • Plate inserts are precoated with ECM basement membrane
Plattentyp Pre-coated
Protokoll The CytoSelect™ Cell Invasion Assay Kit contains polycarbonate membrane inserts (8 μm pore size) in a 24-well plate. The upper surface of the insert membrane is coated with a uniform layer of dried basement membrane matrix solution. This basement membrane layer serves as a barrier to discriminate invasive cells from non-invasive cells. Invasive cells are able to degrade the matrix proteins in the layer, and ultimately pass through the pores of the polycarbonate membrane. Finally, the cells are removed from the top of the membrane and the invaded cells are stained and quantified.
Testdurchführung
  1. Under sterile conditions, allow the invasion chamber plate to warm up at room temperature for 10 minutes.
  2. Rehydrate the basement membrane layer of the cell culture inserts by adding 300 μL of warm, serum-free media to the inner compartment. Incubate at room temperature for 1 hour.
  3. Prepare a cell suspension containing 0.5-1.0 x 106 cells/mL in serum free media. Agents that inhibit or stimulate cell invasion can be added directly to the cell suspension. Note: Overnight starvation may be performed prior to running the assay
  4. Carefully remove the rehydration medium (step 2) from the inserts without disturbing the basement membrane layer. Note: It will not affect the assay performance if a small amount of rehydration medium is left in the compartment
  5. Add 500 μL of media containing 10 % fetal bovine serum or desired chemoattractant(s) to the lower well of the invasion plate.
  6. Add 300 μL of the cell suspension solution to the inside of each insert.
  7. Incubate for 24-48 hours at 37 °C in 5 % CO2 atmosphere.
  8. Carefully aspirate the media from the inside of the insert. Wet the ends of 2-3 cotton-tipped swabs with water, flatten the ends of the swabs by pressing them against a clean hard surface, and gently swab the interior of the inserts to remove non-invasive cells. Take care not to puncture the polycarbonate membrane. Be sure to remove cells on the inside perimeter of the insert.
  9. Transfer the insert to a clean well containing 400 μL of Cell Stain Solution and incubate for 10 minutes at room temperature.
  10. Gently wash the stained inserts several times in a beaker of water. Allow the inserts to air dry.
  11. (optional) Count invasive cells with a light microscope under high magnification objective, with at least three individual fields per insert.
  12. Transfer each insert to an empty well, adding 200 μL of Extraction Solution per well, then incubating 10 minutes on an orbital shaker.
  13. Transfer 100 μL from each sample to a 96-well microtiter plate and measure the OD 560nm in a plate reader. 4
Beschränkungen Nur für Forschungszwecke einsetzbar
Lagerung 4 °C
Informationen zur Lagerung Store all components at 4°C.
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