Cell Invasion Assay kit (24-well plate, 8μM)

Self-brought instruments, reagents and consumables:
Invasive cell line
Cell culture medium with 10% fetal calf serum
Serum-free medium (i.e. cell culture medium with 0% fetal bovine serum)
Cell incubator (37 ℃, 5% CO₂）
PBS, cotton swab, forceps, 96-well plate, 24-well plate, adjustable pipette gun and tip
Microscope
Microplate reader (capable of measuring absorbance at 570 nm)

Reagent Preparation:
24-Well Invasion Plate: ready-to-use; Before use, equilibrate to room temperature; Store at 4 °C.
Matrigel: thaw at 4 °C; Place it on ice before use, and store the unused reagents at-80 ℃ in divided packs.
Fixation Solution: ready to use; Before use, equilibrate to room temperature; Store at-20 °C.
Staining Solution: ready to use; Before use, equilibrate to room temperature; Store at 4 °C.
Elution Solution: Ready-to-use; Before use, equilibrate to room temperature; Store at 4 °C.

Experimental procedure:
Note: Before the formal experiment, a pre-experiment must be conducted to determine the best experimental conditions for cell invasion. Cells must have the ability to secrete matrix metalloproteinases (MMPs) in order to undergo cell invasion. It is recommended to conduct a qPCR pre-experiment to detect the expression of MMPs, especially MMP-2, before the experiment.
1. Prepare Matrigel: Put the gun tip, gun tip box, EP tube, serum-free medium and 24-Well Invasion Plate into the refrigerator for pre-cooling. The Matrigel was removed from − 80 °C, and the Matrigel was thawed at 4 °C. After thawing, the Matrigel was placed in an ice box for operation experiment.
Note: Before the Matrigel forms a gel, mix well with a pre-cooled sterile tip and the Matrigel melts into a clear liquid.
2. Dilute Matrigel: Dilute Matrigel with pre-cooled serum-free medium in an ultra-clean workbench.
Note: (1) Select the appropriate dilution ratio of Matrigel according to the experimental conditions. The dilution ratio setting range is Matrigel: serum-free medium = 1: 6-1: 8, and the recommended dilution ratio is 1: 6. (2) It is not recommended to keep unused Matrigel after dilution for reuse.
3. Please draw 45 µ L of diluted Matrigel from the pre-cooled sterile tip, add it to the upper chamber of 24-Well Invasion Plate, spread it evenly at the bottom, and put it in the cell incubator (37 ℃, 5% CO₂) for 1 h to polymerize Matrigel into gel. According to the actual effect of gel film formation, the incubation time can be appropriately extended.
Note: Do not produce bubbles during the glue laying process, and lay the glue evenly. Generally, the gel can be incubated for 1 hour. It is difficult to see clearly after the gel, so the next experiment can be done directly.
4. Suck off the excess liquid in the upper chamber, add 100 µ L of serum-free medium to each well, and place it in the incubator for 30 minutes to hydrate the basement membrane.
5. Suck off the liquid in the upper chamber, check that the liquid has not passed through the small chamber and entered the lower chamber, and then seed the cells.
Note: If liquid enters the lower chamber, the experimental results may be inaccurate. It is recommended to re-lay the glue.
6. Dilute the cells to 0.5-5.0 × 10 with serum-free medium⁶Cells/mL to prepare a cell suspension.
Note: It is recommended that cells be starved overnight before cell invasion.
7. Add 600 µ L of culture medium containing 10% fetal bovine serum or the required chemoattractant to the lower chamber, then add the small chamber to wet, and add 100 µ L of cell suspension to the upper chamber.
Note: (1) The solution in the upper and lower chambers must not produce bubbles, so that the invasion will be weakened; (2) Cell status is very critical. Try to choose cells with fewer passages.
8. In the cell incubator (37 ℃, 5% CO₂) for 12-48 h.
Note: Too long culture time may cause some invaded cells to fall off from the bottom surface of the membrane. It is recommended to select the appropriate culture time through pre-experiment.
9. Carefully take out the small chamber, suck out the culture medium in the upper chamber, and gently wipe off the uninvaded cells in the upper chamber with the end of a wet cotton swab.
Note: Wipe off the uninvaded cells in the upper chamber. The movement must be gentle and do not puncture the polycarbonate membrane.
10. Transfer the chamber to a well containing 600 µ L of Fixation Solution, fix at room temperature for 10 min, and wash 3 times with PBS.
11. Transfer the chamber to a well containing 600 µ L Staining Solution, stain at room temperature for 5-15 min, wash 3 times with PBS for 3 min each time, and air dry. (If the chamber is still dark stained, it is recommended to gently wash the chamber 3 times in a beaker filled with PBS and dry).
Note: Both ambient temperature and reagent temperature will affect the dyeing time, but a shorter dyeing time will only affect the depth of coloring. If the color is lighter, the dyeing time can be properly heated or extended to achieve the required dyeing effect. Dip dyeing at 37 ℃ for 15 minutes can ensure full coloring.
12. Place the chamber in a clean hole, select at least 3 visual fields with the microscope to observe and take pictures, and calculate the number of invading cells.
13. After taking photos, add 400 µ L of Elution Solution to the lower chamber, elute for 10 minutes, completely elute the Staining Solution, aspirate 200 µ L of the eluted solution into a 96-well plate, and measure the OD value at 570 nm.
Note: (1) Usually, it takes 10 minutes to complete the elution at a room temperature of 22-28 °C. The elution time can be appropriately adjusted according to the temperature difference. (2) You can choose staining photos and measure OD values according to specific experimental needs.

Cell invasiveness refers to the characteristic that cells are stimulated by foreign signals and invade from one place to another. It usually occurs in processes such as wound healing, cell differentiation, embryonic development and tumor metastasis. Transwell is an experimental technology that can simulate many mucosal and biological barrier systems in vitro. The main material of this technology is Transwell chamber. The cell invasion analysis kit (24-well plate, 8 µ m) uses polycarbonate membrane (8 µ m pore size) to determine the invasion characteristics of cells. Matrigel (Matrigel) is the basement membrane matrix extracted from EHS mouse tumors rich in extracellular matrix proteins. Its main components are laminin, type IV collagen, nestin, heparin glycoprotein, and also contains growth factors and matrix metalloproteinases. The principle is to put the Transwell chamber into the culture plate, the chamber is called the upper chamber, and the culture plate is called the lower chamber. The upper and lower layers of culture medium are separated by a polycarbonate membrane, and the matrigel is laid on the upper chamber of the membrane to simulate the extracellular matrix in vivo. If the cells in the upper chamber are to be transferred to the lower chamber, matrix metalloproteinases (MMPs) need to be secreted to degrade the matrigel. Because of the permeability of the polycarbonate membrane, the components in the lower culture medium can affect the cells in the upper chamber. By staining and elution, the number of cells is counted to reflect the invasive ability of cells.

Product Components:

Own instruments, reagents, consumables:
Invasive cell lines
Cell culture medium with 10% fetal bovine serum
serum-free medium (i.e. cell culture medium with 0% fetal bovine serum)
Cell culture incubator (37 ° C, 5% CO₂
PBS
, cotton swabs, forceps, 96 well plate, 24 well plate, adjustable pipet gun and tip
microscope
microplate reader (can measure the absorbance at 570 nm)

reagent preparation:
24-Well Invasion Plate: ready-use type; Before use, balance to room temperature; Store at 4 ° C.
Matrigel: Thaw at 4 ° C; Place on ice before use, and unusable reagents are stored in aliquots at -80 ° C.
Fixation Solution: ready-to-use type; Before use, balance to room temperature; Store at -20 ° C.
Staining Solution: ready-to-use; Before use, balance to room temperature; Store at 4 ° C.
Elution Solution: ready-to-use type; Before use, equilibrate to room temperature; Store at 4 ° C.

Experimental procedure:
Note: Before the formal experiment, it is important to perform a pre-experiment to determine the optimal experimental conditions for cell invasion. Cells must have the ability to secrete matrix metalloproteinases (MMPs) in order to carry out cell invasion. It is recommended to perform qPCR pre-experiments to detect the expression of MMPs, especially MMP-2, before experiments.
1. Prepare the matrix glue: Place the gun tip, gun tip box, EP tube, serum-free medium and 24-Well Invasion Plate in the refrigerator for pre-cooling. The Matrigel was removed from -80℃ and thawed at 4℃. After thawing, the Matrigel was placed in an ice box to operate the experiment.
Note: Before the formation of Matrigel gel, mix the whole process with a precooled sterile tip. After the Matrigel melts, it is a clear liquid.
2. Dilute Matrigel: Dilute Matrigel with pre-cooled serum-free medium in a super-clean bench.
Note: (1) Select the appropriate dilution ratio of Matrigel according to the experimental conditions. The dilution ratio is set to Matrigel: serum-free medium =1:6-1:8, and the recommended dilution ratio is 1:6. (2) The unused Matrigel after dilution is not recommended to be retained for reuse.
3, precooled sterile gun tip suction 45 µ The diluted Matrigel was added to the upper chamber of 24-Well Invasion Plate, evenly spread on the bottom, and placed in the cell culture incubator (37℃, 5% CO₂) for 1 h to polymerize Matrigel into a gel. According to the actual effect of gel film formation, The incubation time can be extended appropriately.
Note: Do not produce bubbles during the spreading process, spreading the glue evenly. Generally, the gel can be incubated for 1 h. It is difficult to see after the gel, so the next experiment can be done directly.
4. Absorb the excess liquid in the upper chamber and add 100&micro to each well; After L serum-free medium, the cells were placed in the incubator for 30 min, and the basement membrane was hydrated.
5, the liquid in the upper chamber is sucked out, and after checking that the liquid does not pass through the chamber into the lower chamber, the cells are inoculated.
Note: If there is liquid into the lower chamber, the experimental results may not be accurate, it is recommended to re-spread the glue.
6. Dilute the cells to 0.5-5.0&times with serum-free medium; 10⁶cells /mL, cell suspension was prepared.
Note: Cells are recommended to be starved overnight before cell invasion.
7. Add 600 &micro to the lower chamber; L medium containing 10% fetal bovine serum or the desired chemoattractant, then add chamber wetting, add 100 &micro to the upper chamber; L of cell suspension.
Note: (1) The solution of the upper and lower chambers must not produce bubbles, so that the invasion effect will be weakened; (2) The state of cells is critical, try to choose cells with fewer passages.
8. The cells were cultured in a cell incubator (37℃, 5% CO₂) for 12-48 hours.
Note: Too long culture time may cause some invasive cells to fall off from the bottom surface of the membrane, so it is recommended to choose the appropriate culture time by pre-experiment.
9, carefully remove the chamber, suck out the medium in the upper chamber, and gently wipe off the non-invaded cells in the upper chamber with the end of a wet cotton swab.
Note: To wipe off the non-invaded cells in the upper chamber, the action must be gentle and do not Pierce the polycarbonate membrane.
10, transfer the chamber to contain 600 µ L Fixation Solution hole, fixed 10 min at room temperature & have spent , PBS & have spent Wash 3 times.
11, to transfer the small room to contain 600 & micro; L Staining Solution Wells were stained at room temperature for 5-15 min, washed 3 times with PBS for 3 min each, and allowed to dry. (if the small room still has deep color, suggested in a beaker of PBS gently cleaning chamber 3 & have spent Allow to dry).
note: the environment temperature and reagent temperature will affect the time needed for dyeing, but short dyeing time affect the depth of the color only, if the color shallow but appropriate heating or extend the dyeing time to achieve the required effect, see at 37 ℃ for 15 min ensures full color.
12, the chamber was placed in a clean well, and at least 3 fields of view were selected for observation and photography under the microscope, and the number of invasive cells was calculated.
13, after taking photos, add 400 &micro to the lower chamber; L Elution Solution and Elution 10 min, the Staining Solution Elution down completely, absorbs the Elution Solution after 200 & micro; L into a 96-well plate and OD was measured at 570 nm.
note: (1) usually in 22 to 28 ℃ under room temperature environment, need 10 min to complete elution, can be adjusted according to the temperature difference appropriate elution time. (2) The staining can be selected to take photos and measure OD value according to the specific experimental requirements.

Store it separately according to the prompt of each component label, and the validity period is 6 months.