肿瘤侵袭试剂盒，24-Well Cell Invasion Assay

Introduction

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.

Cell Biolabs 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.

Assay Principle

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 ultimay 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.

1.   CBA-100: CytoSelect™ 24-Well Cell Migration Assay （8µm, Colorimetric）

2.   CBA-100-C: CytoSelect™ 24-Well Cell Migration and Invasion Assay （8µm, Colorimetric）

3.   CBA-110-COL: CytoSelect™ 24-Well Cell Invasion Assay （Collagen I, Colorimetric）

4.   CBA-110-LN: CytoSelect™ 24-Well Cell Invasion Assay （Laminin I, Colorimetric）

5.   CBA-111: CytoSelect™ 24-Well Cell Invasion Assay （Basement Membrane, Fluorometric）

6.   CBA-111-COL: CytoSelect™ 24-Well Cell Invasion Assay （Collagen I, Fluorometric）

7.   CBA-111-LN: CytoSelect™ 24-Well Cell Invasion Assay （Laminin I, Fluorometric）

8.   CBA-112: CytoSelect™ 96-Well Cell Invasion Assay （Basement Membrane, Fluorometric）

9.   CBA-130: CytoSelect™ 96-Well Cell Transformation Assay （Soft Agar Colony Formation）

Kit Components

1.   ECM Invasion Chamber Plate （Part No. 11001）: One 24-well plate containing 12 ECM-coated cell

culture inserts.

2.   Cell Stain Solution （Part No. 11002）: One Bottle – 10.0 mL

3.   Extraction Solution （Part No. 11003）: One Bottle – 10.0 mL

4.   Cotton Swabs: （Part No. 11004） 40 each.

5.   Forceps: （Part No. 11005） One each. 

Materials Not Supplied

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^ºC, 5% CO2 atmosphere）

5.   Light microscope

6.   96-well microtiter plate

7.   Microtiter plate reader

Storage

Store all components at 4ºC until their expiration dates.

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Assay Protocol

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 37ºC in 5% CO₂ atmosphere. 

8.   Carefully aspirate the media from the inside of the insert.  Wet the ends of 2-3 cotton-tipped

swabs 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.

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Example of Results

The following figures demonstrate typical invasion results with the CytoSelect™ Cell Invasion Assay

Kit.  One should use the data below for reference only.  This data should not be used to interpret actual

results.

Figure 1.  Human Fibrosarcoma HT-1080 Cell Invasion.  HT-1080 and NIH3T3 （negative control）

were seeded at 300,000 cells/well and allowed to invade toward 10% FBS for 24 hrs in the presence or

absence of 2 µM Cytochalasin D.  Invasive cells on the bottom of the invasion membrane were stained

（top panel picture） and quantified at OD 560nm after extraction （bottom panel figure）.   

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.

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