Product Manual

CytoSelect™ 96-Well Cell Invasion Assay(Collagen I, Fluorometric Format)
Catalog Number
CBA-112-COL

96 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
The ability of malignant tumor cells to invade normal surrounding tissue contributes in large part to thesignificant 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.
Cell Biolabs CytoSelect™ 96-well Collagen Cell Invasion Assay Kit utilizes Bovine Type I Collagencoated 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 thequantitative determination of cell invasion. It contains sufficient reagents for the evaluation of 96
samples.

Assay Principle
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 todiscriminate invasive cells from non-invasive cells. Invasive cells are able to degrade the collagenmatrix layer, and ultimately pass through the pores of the polycarbonate membrane. Finally, theseinvaded cells are then dissociated from the membrane and subsequently detected by the patented
CyQuant® GR Dye (Invitrogen).

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Related Products
1. CBA-101-C: CytoSelect™ 24-Well Cell Migration and Invasion Assay (8µm, Fluorometric)
2. CBA-106-C: CytoSelect™ 96- Well Cell Migration and Invasion Assay (8µm, Fluorometric)
3. CBA-111: CytoSelect™ 24-Well Cell Invasion Assay (Basement Membrane, Fluorometric)
4. CBA-111-COL: CytoSelect™ 24-Well Cell Invasion Assay (Collagen I, Fluorometric)
5. CBA-112: CytoSelect™ 96-Well Cell Invasion Assay (Basement Membrane, Fluorometric)

Kit Components
1. 96-well Collagen Invasion Plate (Part No. 111201-COL): One sterile 96-well plate containing
collagen-coated inserts (see Figure 1 for components)
2. 96-well Cell Harvesting Tray (Part No. 10402): One 96-well tray
3. Cell Detachment Solution (Part No. 10403): One 20 mL bottle
4. 4X Lysis Buffer (Part No. 10404): One 10 mL bottle
5. CyQuant® GR Dye (Part No. 10105): One 75 µL tubeMaterials Not Supplied
1.
2.
3.
4.
5.
6.
7.

Invasive cell lines
Cell culture medium
Serum free medium, such as DMEM containing 0.5% BSA, 2 mM CaCl2 and 2 mM MgCl2
Cell culture incubator (37ºC, 5% CO2 atmosphere)
Light microscope
96-well microtiter plate
Microtiter plate reader

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Top

Plate Cover

MiddleInvasion Plate
Membrane Chamber

BottomFeeder Tray

Figure 1: Components of the 96-well Collagen Cell Invasion Plate.

Storage
Store all components at 4ºC.

Assay Protocol
1. Under sterile conditions, allow the collagen invasion plate to warm up at room temperature for 10minutes.
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).
5. Under sterile conditions, separate the cover and membrane chamber from the feeder tray. Add 150µL of media containing 10% fetal bovine serum or desired chemoattractant(s) to the wells of thefeeder tray.
6. Place the membrane chamber back into the feeder tray (containing chemoattractant solution).
Ensure no bubbles are trapped under the membrane.
4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 chamberfrom 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 37ºC.
12. Completely dislodge the cells from the underside of the membrane by gently tilting the membranechamber 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 150µL 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 thefluorescence with a fluorescence plate reader at 480 nm/520 nm.

Example of Results
The following figures demonstrate typical with the CytoSelect™ Collagen Cell Invasion Assay Kit.
One should use the data below for reference only. This data should not be used to interpret actualresults.
350
300

RFU

250
200
150
100
500010000

20000

30000

40000

Cell Number
Figure 2: Quantitation of Human HT-1080. HT-1080 cells were titrated in Cell Detachment Buffer,then subsequently lysed and detected with 4X Lysis Buffer/Cyquant® GR Dye (150µL cell suspension
was mixed with 50µL of 4X Lysis Buffer/dye).

5120100

RFU

800% FBS
10% FBS

6040200NIH3T3HT-1080

Figure 3: Human Fibrosarcoma HT-1080 Collagen Cell Invasion. HT-1080 and NIH3T3(negative control) were seeded at 70,000 cells/well and allowed to invade toward FBS for 24 hrs.
Invasive cells on the bottom of the invasion membrane were quantified by CyQuant® GR dye as
described in the Assay Protocol.

References
1. Erkell, L. J., Schirrmacher, V. (1988) Cancer Res 48, 6933-6937.
2. Montgomery, A. M. P., De Clerck, Y. A., Langley, K. E., Reisfeld, R. A., Mueller, B. M. (1993)
Cancer Res 53,693-700.
3. Monsky, W. L., Lin, C. Y., Aoyama, A., Kelly, T., Akiyama, S. K., Mueller, S. C., Chen, W. T.
(1994) Cancer Res 54,5702-5710.

Recent Product Citations
1. Negro, G. et al. (2020). Molecular heterogeneity in breast carcinoma cells with increased invasivecapacities. Radiol Oncol. 54(1):103‐118. doi: 10.2478/raon-2020-0007.
2. Matsui, A. et al. (2018). Prolonged engraftment of transplanted hepatocytes in the liver by transient
pro-survival factor supplementation using ex vivo mRNA transfection. J Control Release. 285:111. doi: 10.1016/j.jconrel.2018.06.033.
3. Ohishi, T. et al. (2017). Tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton
rearrangement and cancer cell invasion. Cancer Res. 1846.2016. doi: 10.1158/0008-5472.CAN-161846.
4. Ben-David, U. et al. (2016). The landscape of chromosomal aberrations in breast cancer mousemodels reveals driver-specific routes to tumorigenesis. Nat Commun. doi:10.1038/ncomms12160.
65. Ho, P. W. et al. (2015). Knockdown of PTHR1 in osteosarcoma cells decreases invasion andgrowth and increases tumor differentiation in vivo. Oncogene. 34:2922-2933.
6. Ishiba, T. e. al. (2014). Periostin suppression induces decorin secretion leading to reduced breast
cancer cell motility and invasion. Sci Rep. 4:7069.
7. Ziemann, A. et al. (2013). CRN2 enhances the invasiveness of glioblastoma cells. Neuro Oncology
10.1093/neuonc/nos388.
8. Liu, X. et al. (2013). Antiproliferative, antiinvasive, and proapoptotic activity of folate receptor αtargeted liposomal doxorubicin in nonfunctional pituitary adenoma cells. Endocrinology 154:14141423.
9. Ziemann, A. et al. (2013). CRN2 enhances the invasiveness of glioblastoma cells. Neuro Oncology
10.1093/neuonc/nos388.

License Information
This product is provided under an intellectual property license from Life Technologies Corporation.
The purchase of this product conveys to the buyer the non-transferable right to use the purchased
product and components of the product only in research conducted by the buyer (whether the buyer isan academic or for-profit entity). The sale of this product is expressly conditioned on the buyer notusing the product or its components, or any materials made using the product or its components, in any
activity to generate revenue, which may include, but is not limited to use of the product or its
components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment;(iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they
are resold for use in research. For information on purchasing a license to this product for purposes
other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad
CA 92008 USA or outlicensing@lifetech.com.

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In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

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