细胞迁移和浸润实验操作 protocol

For life science research only.
Not for use in diagnostic procedures.
System
Technical Note No. 3 / February 2010 General Cell Migration Protocol Using
the CIM-Plate 16
Cell Migration Assay Using the CIM-Plate 16 with the RTCA DP Instrument
1.
Protocol
Reagents
1.1
᭤HeLa and HT-1080 cells, purchased from ATCC and at 60% – 80% confl uence at the time of detachment T he ultimate success of the migration experiments is dependent upon cell culture conditions prior to the assay and conditions used for detaching the cells from the fl ask. The number of cells used in a migration experiment will ultimately depend on the cell type being used. It is imperative to conduct preliminary experiments and determine the optimal cell number for each cell line. We recommend initially seeding cells in the range of 10,000 – 80,000 cells in a fi nal volume of 100 µl.
᭤T rypsin-EDTA solution for cell detachment, or Non-enzymatic Cell Dissociation Solution (Sigma Cat. No. C5789) for cells that may be especially sensitive to enzymatic methods of cell detachment
I f using the protease method for cell detachment, it is important to minimize the time of incubation with the
detachment solution. Cell surface receptors (such as integrins) play an important role in cell migration and it is important to conserve the number and integrity of these receptors on the cell surface as much as possible.
᭤TNS solution (Clonetics, Cat. No. CC-5002)
᭤Serum-free medium (SFM), i.e., the same media that the cells are cultured in, without the serum
C ertain cell types are sensitive to total absence of serum and it may be necessary to include some serum in the SFM.
We recommend testing a range of 0.1% to 2% serum. Alternatively, the SFM can be supplemented with bovine serum albumin (BSA) in the range of 0.25% to 0.5%. It is important to use highly pure forms of BSA and not the crude fraction product which may contain factors that could affect cell adhesion and migration. We recommend using 30% BSA stock solution from Sigma (Cat. No. A9576).
᭤Phosphate buffered saline (PBS), HyClone (Cat. No. 3002201)
᭤C hemotaxis inducer, typically SFM supplemented with serum in the range of 5% to 10%, conditioned fi broblast media, or SFM supplemented with chemotactic agents such as growth factors
I f low serum or BSA was used as part of the SFM, it may be important to add the chemotactic factor to the same media.
1.2
Equipment
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᭤
Procedure
2.
Day 1
CIM-Plate 16 Assembly
A. Time: approx. 5 minutes
remaining wells (see Figure 1).
E nsure that a meniscus is formed on each well after the well is fi lled with media.
T o prevent bubble entrapment in the LC, do not eject all of the media from the tip when pipeting media. Leave about 0.5 µl in the tip. In the event of bubble formation, try using a dry tip to “pop” the bubble or carefully re-move it using a pipette.
᭤᭤᭤
3Place the UC in one of the unused pockets of the assembly tool in the correct orientation, where the blue marking on the assembly tool lines up with the blue marking on the UC. Turn the assembly tool (do not slide) 90 degrees counter-clockwise and then pick up the UC using both hands and carefully place the UC onto the LC with the sensor surface facing down and the blue marking on the UC matching the blue marking on the LC.
E nsure that the UC and LC are level and parallel to each other as the device is being assembled. Do not tilt the
UC.
4Push the UC onto the LC. Make sure you hear two clicks, indicating that the two chambers are locked together and sealed properly.
L et the UC slide down slowly. Do not push until both chambers are in contact.
5Add 25 - 50 µl (recommended: 30 µl) SFM to the center of each well of the UC to cover the membrane surface. Do not add SFM close to the wall. This may result in an uneven distribution of SFM due to surface tension. Do not introduce any bubbles, and do not allow the pipette tips to touch the membrane.
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B. Time: approx. 60 minutes
C IM-Plate 16 Equilibration at +37°C
1Place the CIM-Plate 16 onto the RTCA DP Analyzer inside the incubator at +37˚C. Make sure the tapered corner on the CIM-Plate 16 matches the tapered corner on the RTCA DP Analyzer. Incubate for 1 hour to allow the membrane surface to reach equilibrium with the media.
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C. Time: approx. 5 minutes
Background Measurement
1 In the RTCA Software, start Step 1 (1 minute and 1 sweep) to perform background measurement.
■D. Time: approx. 20 minutes
Cell Preparation
L ike any other cell-based assay, the ultimate success of a migration experiment using the CIM-Plate 16 depends on the quality and handling of the cells. It is imperative to critically review and follow the steps for cell maintenance and handling in order to ensure reliable and reproducible results. Also, it is important to take note of the passage number of the cells, as certain cell types can attain higher levels of background migration with increasing passage number.
1Cells should be passaged the day prior to the experiment and should be 60 – 80% confl uent.
S ome cell types may possess an inherently high level of background migration; it may be important to serum-
starve the cells prior to detachment. Cells can be serum-starved for 1 to 16 hours prior to detachment for migration
assay. These conditions need to be determined empirically.
2Remove serum-containing medium from the fl ask and gently rinse cell monolayer once with PBS.
3Trypsinize cells by adding 0.5 ml of 0.05% Trypsin/EDTA solution per T-25 fl ask and leave the fl ask at +15 to +25°C or +37°C for 1 – 2 minutes. Do not over-trypsinize the cells. Check degree of trypsinization under a microscope.
D o not over-trypsinize the cells: Cell migration and invasion are dependent on the expression and integrity of cell
surface receptors such as integrins; it is important to minimize the time of protease treatment. Certain cell types
can be sensitive to protease digestion, and it may be important to explore alternative methods of cell detachment
(i.e., using EDTA-based buffers).
D o not leave the detached and suspended cells on ice. This incubation can cause aggregation and therefore
impact migration potential of the cells.
4Stop trypsinization by adding low-serum media or TNS solution (Clonetics, Cat. No. CC-5002) at a ratio of 1:1.
5Wash trypsinized cells once with SFM by centrifugation. For most cells, 5 minutes at 800 × g is suffi cient.
6Gently resuspend the cell pellet in a few milliliters of SFM and count the cells under a microscope using a hemo c ytometer or an automated counter. Adjust the concentrations of multiple aliquots of the cell suspension to 4 × 105 cells/ml and 2 × 105 cells/ml, using SFM; use at least triplicates or quadruplicates for each experimental condition.
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D uring addition of the cells the pipette tip should touch the surface of media already present in the well.
Figure 1: Arrangement of two cell concentrations (4 × 105 cells/ml and 2 × 105 cells/ml) in the upper chamber (UC) of the CIM-Plate 16, and arrangement of attractant and control media in the lower chamber (LC) of the CIM-Plate 16.
B e aware that bubbles can be entrapped in the bottom of the well, forcing cells to migrate at the edge or block
cell migration altogether. The best way to avoid bubble entrapment is to fi rst slightly tilt the device at an angle,
followed by insertion of the pipette tips all the way to the bottom of the well of the UC (avoiding the electrodes)
and slowly ejecting the cell suspension. One way to check for possible bubble entrapment is to observe the level
of the medium in the wells of the UC. If you added the same volume to each well, then the volume level in all
the wells should be the same. If any wells contain air pockets or bubbles, then the volume level will be slightly
higher than in the other wells. If you observe this immediately after addition of cell suspension, you can carefully
aspirate the cell suspension and reapply it to the well. However, if this is observed later, it is better to avoid
removal of the bubble and simply make a note of it in your laboratory notebook.
■F. Time: approx. 30 minutes
C IM-Plate 16 Equilibration at Room T emperature
1Leave the CIM-Plate 16 in the tissue culture hood at +15 to +25°C for 30 minutes after cell addition to allow the cells to settle onto the bottom surface of the UC.
■G. Time: approx. 6 – 24 hours
Start Measurement
1Place the CIM-Plate 16 containing the cells in the RTCA DP Analyzer inside the incubator. Take measurements for 24 hours at intervals of 15 minutes.
cient of variation of the
to choose a time point that is well below the doubling time of the specifi c cell type in order to avoid complications
Day 2
H.
Data Analysis
1Stop the measurement after 24 hours.
■3.
Typical Results
Both HT-1080 and HeLa cell lines possess strong cell migration potential. Migration activities of the two cell lines were tested on the RTCA DP Instrument using 10% FBS as an attractant. The cell migration kinetics for initial seeding density of 20,000 and 40,000 cells/well was recorded in real time (Figure 2, A and B). The migration CIs of HT-1080 cells and HeLa cells were correlated with inputted cell numbers of each cell line. Figure 2 panels C and D show microscopic images of the stained HT-1080 and HeLa cells for UC wells initially seeded with 20,000 cells, after the cell migration experiment was stopped at 20 hours.
A B
C D
Figure 2: (A and B) Migration kinetics of HT-1080 cells and Hela cells by continuous monitoring of live cell migration for approximately 22 hours. Red: 40,000 cells/well, 10% FBS in LC, n = 6; green: 20,000 cells/well, 10% FBS in LC, n = 6. Negative controls: Blue: 40,000 cells/well, SFM in LC, n = 2; magenta: 20,000 cells/well, SFM in LC, n = 2; error bars represent standard deviation. (C and D) Representative images of migrated HT-1080 and Hela cells, respectively, stained with Diff Quick Staining Kit on the underside of the membrane at the end of the experiment. Initial seeding number: 20,000 cells/well.
Appendix A: Staining
Even though not required, it is recommended when performing pilot experiments to stain the cells on the underside of the membrane to get an impression of the extent of cell migration and its correlation with the CI signal. Diff Quick Staining Kit (Fisher Scientifi c; Cat. No. NC9409820) is recommended. The modifi ed staining procedure is briefl y described below:
᭤Disassemble the CIM-Plate 16 by pressing the two handles on the LC and gently removing the UC.
᭤Remove media from wells of the UC with a multi-channel pipette while avoiding contact with the membrane.
᭤Remove cells from the upper side of the membrane, e.g., using a cotton swab.
᭤S tain the migrated cells on the bottom side of the membrane: Place the UC into fi xative solution for 1 - 2 minutes with membrane side (containing the electrodes) in direct contact with the fi xative.
᭤Place the UC into solution I for 0.5 - 1 minute with membrane side in direct contact with the solution.
᭤Place the UC into solution II for 0.5 - 1 minute with membrane side in direct contact with the solution.
᭤Gently rinse the UC with water.
᭤Visualize and count the cells under an upright microscope.
Appendix B: Troubleshooting
This protocol was developed for HT-1080 and HeL a cell lines. For cell lines with low migration potential, further optimization is needed to achieve a better signal. The main factors affecting the quality and reproducibility of the data are general cell quality, possible entrapment of air pockets or bubbles, and improper sealing of the UC and LC. It is important to pay particular attention to these details.
1. No signal or lower signal than usual:
᭤Bubbles may be trapped in the UC wells or LC wells.
᭤Cell counting or cell seeding number may be incorrect.
᭤C ells may be unhealthy due to contamination, over-trypsinization, or over-confluence at the point of trypsinization.
᭤Cells may be unhealthy due to inappropriate serum starvation.
᭤ CO
2 and temperature of the CO
2
incubator may be inappropriately set.
᭤The chemoattractant used may be degraded or not optimal for the cell type of interest.
᭤Wells of the CIM-Plate 16 may be contaminated.
᭤Medium leakage may occur due to improper assembly of the UC and LC.
2. High well-to-well variability:
᭤I nitial seeding of the cells may be inconsistent due to poor mixing of the cells or due to clumps of cells present in suspension.
᭤Membranes may have been damaged by pipette tips during cell or medium addition.
᭤The incubation step (holding the cells at +15 to +25°C for 30 minutes) was not followed.
᭤The cells may have been kept on ice upon detachment.
᭤Variations in accuracy of pipettes or pipette handling may contribute to well-to-well variability.
᭤Bubbles may have been trapped in the wells.
᭤Media leakage may have occurred.
3. Curve fl uctuation and/or delayed curve:
᭤The contact pads may be short circuited by media due to over-fi lling of the lower chamber wells.
᭤Media leakage may have occurred during the measurement.
Intended Use:
For life science research only. Not for use in diagnostic procedures.
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