CytoSelect™ 96-well Collagen Cell Invasion Assay, Fluorometric

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Reactivity
Mammalian
Application
Cellular Assay (CA)
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Purpose The CytoSelect™ 96-well Collagen Cell Invasion Assay Kit contains polycarbonate membrane inserts (8 μm pore size) in a 96-well plate. The upper surface of the insert membrane is coated with a uniform layer of dried Bovine Type I Collagen matrix. This collagen matrix layer serves as a barrier to discriminate invasive cells from non-invasive cells. Invasive cells are able to degrade the collagen matrix layer, and ultimately pass through the pores of the polycarbonate membrane. Finally, these invaded cells are then dissociated from the membrane and subsequently detected by the patented CyQuant® GR Dye (Invitrogen). 2
Brand CytoSelect™
Sample Type Serum, Cell Samples
Analytical Method Quantitative
Detection Method Fluorometric
Characteristics CytoSelect™ 96-well Collagen Cell Invasion Assay Kit utilizes Bovine Type I Collagen- coated inserts to assay the invasive properties of tumor cells. The kit does not require you to prelabel the cells with Calcein AM or remove non-invaded cells (i.e. cotton swabbing). Any invaded cells are first dissociated from the membrane, then lysed and detected with CyQuant® GR Dye. The CytoSelect™ 96-well Collagen Cell Invasion Assay Kit provides a robust system for the quantitative determination of cell invasion. It contains sufficient reagents for the evaluation of 96 samples.
Components
  1. 96-well Collagen Invasion Plate : One sterile 96-well plate containing collagen-coated inserts (see Figure 1 for components)
  2. 96-well Cell Harvesting Tray : One 96-well tray
  3. Cell Detachment Solution : One 20 mL bottle
  4. 4X Lysis Buffer : One 10 mL bottle
  5. CyQuant® GR Dye : One 75 μL tube
Material not included
  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 3 Top Plate Cover Middle Invasion Plate Membrane Chamber Bottom Feeder Tray : Components of the 96-well Collagen Cell Invasion Plate.
Background 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.
Application Notes Optimal working dilution should be determined by the investigator.
Comment

  • Fully quantify cell invasion with no manual cell counting
  • Plate inserts are precoated with Collagen I gel layer

Plate Pre-coated
Assay Procedure
  1. Under sterile conditions, allow the collagen invasion plate to warm up at room temperature for 10 minutes.
  2. Rehydrate the collagen layer of the membrane inserts by adding 125 μL of warm, serum-free media to the inner compartment. Incubate at room temperature for 30 minutes.
  3. Prepare a cell suspension containing 0.2-2.0 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 100 μL of the rehydration medium (step 2) from the inserts without disturbing the collagen layer (leaving 25 μL inside). 4
  5. Under sterile conditions, separate the cover and membrane chamber from the feeder tray. Add 150 μL of media containing 10 % fetal bovine serum or desired chemoattractant(s) to the wells of the feeder tray.
  6. Place the membrane chamber back into the feeder tray (containing chemoattractant solution). Ensure no bubbles are trapped under the membrane.
  7. Gently mix the cell suspension from step 3 and add 100 μL to the membrane chamber.
  8. Finally, cover the plate and transfer to a cell culture incubator for 12-24 hours.
  9. Just prior to the end of the incubation, pipette 150 μL of prewarmed Cell Detachment Solution into wells of the clean, 96-Well Cell Harvesting Tray (provided).
  10. Carefully remove the 96-well Invasion Plate from the incubator. Separate the membrane chamber from the feeder tray.
  11. Remove the cells/media from the top side of the membrane chamber by aspirating or inverting. Place the membrane chamber into the Cell Harvesting Tray containing 150 μL of Cell Detachment Solution (step 9). Incubate 30 minutes at 37 °C.
  12. Completely dislodge the cells from the underside of the membrane by gently tilting the membrane chamber several times in the Cell Detachment Solution.
  13. 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).
  14. Add 50 μL of 4X Lysis Buffer/CyQuant® GR dye solution to each well (already containing 150 μL of Cell Detachment Solution). Incubate 20 minutes at room temperature.
  15. Transfer 150 μL of the mixture to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorescence plate reader at 480 nm/520 nm. 5
Restrictions For Research Use only
Storage 4 °C
Storage Comment Store all components at 4°C.
Product cited in: Ohishi, Yoshida, Katori, Migita, Muramatsu, Miyake, Ishikawa, Saiura, Iemura, Natsume, Seimiya: "Tankyrase-Binding Protein TNKS1BP1 Regulates Actin Cytoskeleton Rearrangement and Cancer Cell Invasion." in: Cancer research, Vol. 77, Issue 9, pp. 2328-2338, 2017 (PubMed).

Ben-David, Ha, Khadka, Jin, Wong, Franke, Golub: "The landscape of chromosomal aberrations in breast cancer mouse models reveals driver-specific routes to tumorigenesis." in: Nature communications, Vol. 7, pp. 12160, 2016 (PubMed).

Ho, Goradia, Russell, Chalk, Milley, Baker, Danks, Slavin, Walia, Crimeen-Irwin, Dickins, Martin, Walkley: "Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo." in: Oncogene, Vol. 34, Issue 22, pp. 2922-33, 2015 (PubMed).

Ishiba, Nagahara, Nakagawa, Sato, Ishikawa, Uetake, Sugihara, Miki, Nakanishi: "Periostin suppression induces decorin secretion leading to reduced breast cancer cell motility and invasion." in: Scientific reports, Vol. 4, pp. 7069, 2014 (PubMed).

Liu, Ma, Dai, Cai, Yao, Yang, Feng, Deng, Li, Ma, Xin, Lian, Xiang, Zhang, Wang: "Antiproliferative, antiinvasive, and proapoptotic activity of folate receptor α-targeted liposomal doxorubicin in nonfunctional pituitary adenoma cells." in: Endocrinology, Vol. 154, Issue 4, pp. 1414-23, 2013 (PubMed).

Ziemann, Hess, Bhuwania, Linder, Kloppenburg, Noegel, Clemen: "CRN2 enhances the invasiveness of glioblastoma cells." in: Neuro-oncology, Vol. 15, Issue 5, pp. 548-61, 2013 (PubMed).