发帖
每发1个新帖
可以获得0.5个丁当奖励
回帖

细胞侵袭试验的Protocol

楼主

丁香园荣誉版主

1楼

这个帖子发布于15年零219天前，其中的信息可能已发生改变或有所发展。

从很早以前就有园友以遍又一遍地问用Transwell做细胞侵袭试验的问题和Transwell的用途问题，对于后一个问题，实在没必要讲，我曾给我带的学生讲了一下午Transwell的各种用法，实在太累。
不过侵袭试验倒是可以发个Protocol
Invasion Assays

To metastasize, tumor cells invade through vessel basement membrane and the underlying connective tissue by attaching to, degrading, and migrating across the basement membrane matrix (Liotta, 1989). The in vitro assay described in this unit is performed to determine the invasive character of tumor cells, to test for factors that promote or inhibit invasion, and to select for malignant subpopulations, which are invasive (Kramer et al., 1986; Albini et al., 1987). The assay is rapid, quantitative, and proven to be highly reliable in a number of research laboratories.

Matrigel is a better barrier than amnion or other natural substrates, because it shows high reproducibility in its activity, and the assay time is much shorter (Hendrix et al., 1989). Many known inhibitors of tumor metastases, such as protease inhibitors, block invasion in this assay (Reich et al., 1988; Stahl and Meuller, 1994; Kobayashi et al., 1995). Likewise certain growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor  (TGF), hepatocyte growth factor (HGF), and thyroid-stimulating hormone (TSH), have all been found to promote invasion (Nicolson et al., 1993; Hoelting et al., 1994; Rong et al., 1994; Sunitha et al., 1994; Chambers et al., 1995). Having an assay that is quick, reliable, and accurate can facilitate diagnosis and aid in the development of therapeutics. The assay can be used as a prescreen for a large number of test compounds.

Critical Parameters and Troubleshooting

The exact time of incubation varies depending on the cells used and the amount of coated Matrigel. Trials using different incubation time in the presence and absence of a chemoattractant such as 3T3-conditioned medium should be performed initially to determine the optimum time of incubation. The optimum is the time at which there is the largest difference in the number of infiltrating cells between the control and positive attractant. In some cases, it may also be desirable to vary the number of cells added. For example, if the cells are highly invasive and too numerous to count, fewer cells can be used. In addition, because of the assay's sensitivity, if possible stimulators or inhibitors are being assessed, several different concentrations of the test substance should be tested.

Choosing an appropriate attractant is important. Many studies employ conditioned medium from 3T3 cells, which is collected for 24 hr with serum-free medium containing 50 ug/ml ascorbic acid. The conditioned medium is generally used directly without preconcentration. With cells that have not been previously tested, it may be desirable to test a range of concentrations. Several other chemoattractants have also been identified and include various growth factors, such as EGF (Hoelting et al., 1995), bFGF (Hasegawa et al., 1994), and HGF (Rong et al., 1994). Tissue extracts have been found to act as chemoattractants with some cell types (Hujanen and Terranova, 1985), and laminin as well as other matrix molecules can also promote invasion when placed in the lower well of the chamber (Koochepour et al., 1995). To determine the best chemoattractant, it is advisable to test 3T3-conditioned medium first and then various growth factors and matrix molecules if needed. Cells generally respond by migrating toward growth factors for which they have receptors. The attractant needs to be in the lower well of the chamber and should be present during the entire assay. It may be necessary to test various concentrations as cells usually migrate to factors at concentrations approximately one-tenth of that needed for growth, and many chemoattractants are inhibitory for migration when used at high concentrations. Preincubation with the chemoattractant is not required.

It is important to include controls in each assay. Generally the negative control is medium alone (lacking attractant), whereas the positive control is either 3T3-conditioned medium or a medium containing a substance already identified as a chemoattractant for the cells being tested. The assay should be performed in triplicate for the best results.

Anticipated Results

Results are assessed quantitatively by comparing the cell counts for experimental and control wells or plates: for example, when testing an attractant, very few cells invade in its absence, whereas more cells (usually at least twice background) should be observed in its presence.

Time Considerations

The assay should be completed in 1 day. Filters can be coated the night before the assay. Once fixed, the filters are stable for months and can be easily stored in slide boxes.

INTRODUCTION

The basement membrane is a thin extracellular matrix that underlies epithelial and endothelial cells and separates these tissues from stroma. Tumor cells must cross the vessel basement membrane and penetrate the underlying stroma in order to invade tissue and form distant metastases. They do this by producing proteases that degrade the extracellular matrix. Several in vitro invasion assays have been developed using various extracellular matrix barriers including amnion, type I collagen gels, and a reconstituted basement membrane termed Matrigel (see UNITS 10.2 & 10.3). Matrigel-based invasion assays (see Basic Protocol) are the most reliable, reproducible, and representative of in vivo events, and they are the assays most frequently used. Porous filters are coated with a thin layer of Matrigel and placed in a Boyden migration chamber with a chemoattractant in the lower well and tumor cells in the upper well (Fig. 12.2.1). The entire chamber is then incubated for ~3 to 10 hr, depending on the tumor cells used. After incubation, the filter is removed, fixed, and stained, and the cells on the lower surface of the filter are quantitated. This assay is advantageous because it is quick, reliable, quantitative, and in most cases does not require sterility. Compounds that either promote or inhibit invasion can be assayed. In addition, at the end of the assay, the invasive cells can be recovered and used for further study. The invasion assay can be used to screen for a large variety of compounds in 48-well chambers, with the advantages that smaller amounts of test material and fewer cells are needed. Commercial kits are also available. This protocol is provided for those investigators who seek to test a large number of samples or to modify the basic protocol (for example, by increasing or decreasing the amount of Matrigel coated on to accommodate cells with high or low invasive activity, respectively).

Figure 12.2.1 Configuration of chamber used for invasion assay. (A) Schematic representation of the Boyden chamber assembly. (B) Photomicrograph of matrix barrier cross-section, consisting of a porous polycarbonate filter coated with Matrigel. Reproduced with permission from the American Association for Cancer Research (Albini et al., 1987).

BASIC PROTOCOL: MEASURING INVASION THROUGH A MATRIX

This protocol describes a method for assessing tumor cell invasion through a basement membrane matrix in vitro. Tumor cells are placed in the upper well of a Boyden migration chamber, which is separated from the lower well by a porous filter coated with basement membrane matrix (Matrigel). A chemoattractant is placed in the lower well to facilitate cell migration. After a short incubation, invasive cells are counted or recovered from the lower surface of the filter.

Materials

3T3-conditioned medium (see recipe) or selected chemoattractant or inhibitor
Tumor cells in culture
0.5 M EDTA, pH 8.0 (APPENDIX 2A)
Culture medium containing 0.1% (w/v) BSA (varies by cell type)
Diff-Quik fixative and stains (Baxter)

Polyvinylpyrrolidone (PVP)-free polycarbonate membranes, 8- or 12-um pore size (e.g., Nucleopore filters, Neuro Probe), sized to fit Boyden chambers (13-mm for single chambers and 25  80-mm for 48-well chambers) coated with Matrigel (see Support Protocol)

1. Add 3T3-conditioned medium or other chemoattractant to be tested to lower wells of Boyden chambers until a small meniscus appears.

Each assay data point is generally based on triplicate filters/wells. Always include three negative control wells that lack the migration factor in the lower well. If using 48-well chambers, each filter should include three positive- and three negative-control wells (i.e., medium alone). Generally nano- or microgram amounts of chemoattractants are used. It is best to test several different concentrations.

2. Place a Matrigel-coated filter over the lower well of each Boyden chamber and secure the filter with a gasket.

For 48-well chambers, start putting the filter down from the middle of the chamber to the edges to avoid bubbles. Never move the filter after it has been placed or there will be a possibility of cross-contamination of the wells. Do not screw the gasket too tight.

3. Release tumor cells from their culture vessel by first removing the culture medium and then adding enough 0.5 M EDTA to fully cover the cell layer in the culture dish. Centrifuge the cell suspension 5 min at 170  g in a tabletop centrifuge, room temperature. Decant the supernatant and resuspend the pellet in serum-free medium at 5  105 cells/ml (1  106 cells/ml for 48-well chambers).

The cells should not be confluent. Best results are obtained when the cells are split 24 hr before the assay and always seeded at the same density. It is very important to use EDTA rather than trypsin to release the cells from the plate, as trypsin will degrade the Matrigel.

4. Add cells to the upper wells of the chambers.

Generally 100,000 to 200,000 cells per 0.3-ml well are placed in the upper chamber. When using the smaller 48-well chambers, 50,000 cells per 0.05-ml well are placed in each upper chamber well.

5. Incubate 3 to 6 hr in a humidified 5% CO2, 37C incubator.

The time of incubation is dependent on the tumor cells used; 3 to 6 hr is the most frequently used range of incubation times. The optimal time can be determined in an initial test assay with and without attractant in the lower well, looking for the greatest difference between the two conditions. Usually <2% of the added cells migrate in the presence of the attractant.

NOTE: All culture incubations should be performed in a humidified 37C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

6. Stop the incubation by removing and fixing the filters using the Diff-Quik system. Remove the filters, dip in fixative for 10 sec, and place in solutions I and II for 2 min each. Wash in water for a few seconds.

Small weigh boats or Coplin jars may be used for these steps and the solutions can be reused. The cells will appear dark blue with a pale blue to white background.

7. Wipe cells from the upper surface with a cotton-tipped swab and mount filter on a slide with the lower side down.

This step removes the cells that have attached to the upper surface of the filter and makes counting easier. The migrated cells will be on the underside of the filter facing the glass slide.

8. Count the cells.

This can be done with a computer system or by eye (see Fig. 12.2.2). Be sure to survey the entire filter, as the distribution of migrated cells may be uneven. Count the most representative areas. Counting with a 10 magnification using a grid has proven to be reliable and reproducible.

Figure 12.2.2 Photomicrographs showing results of an invasion assay: filter underside (attractant side) after incubation with (A) noninvasive and (B) invasive cells. Reproduced with permission from the American Association for Cancer Research (Albini et al., 1987)..

SUPPORT PROTOCOL: PREPARATION OF MATRIGEL-COATED FILTERS

Coating the filter with Matrigel provides a surface similar to that encountered by cells migrating in vivo.

Materials

PVP-free polycarbonate membranes, 8- or 12-um pore size (e.g., Nucleopore filters, Neuro Probe), sized to fit Boyden chambers (13-mm for single chambers; 25  80-mm for 48-well chambers)
Matrigel (UNIT 10.2; Matrigel can also be obtained commercially from Becton Dickinson Labware or Sigma)

1. Number the filters on the dull side with a permanent ink pen.

2a. If using single chambers: Dilute Matrigel to 1 ug/ul and pipet 25 ul as a center spot on the dull side of the 13-mm-diameter filter. Air dry overnight.

2b. If using 48-well chambers: Dilute Matrigel to 0.5 ug/ul in cold water. Place the filter shiny side down and cover the top with 1 ml of the diluted Matrigel. Incubate 15 to 20 min, then hang the filter to air dry for 5 to 10 min (a pin or needle stuck in one corner through the filter and into a board is convenient for drying). Cut the corner so that the orientation of the filter can be determined at the end of the assay.

我们的做法和这个Protocol做了些改动差不多
这是我们的结果：还算就和;-) 可以看到traswell的小孔的穿过的细胞