Cell Migration Assay Kit (24-well plate, 8μM)

Self-brought instruments, reagents and consumables:
Migratory 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 Migration Plate：Ready-to-use; Before use, equilibrate to room temperature; Store at 4 °C.
Fixation：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, be sure to conduct a pre-experiment to determine the best experimental conditions for cell migration, such as cell number, cell culture time and staining time.
1. In an ultra-clean workbench, the 24-well Migration Plate is equilibrated at room temperature for 10 minutes.
2. 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 migration.
3. 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 the migration will be weakened; (2) Cell status is critical, and try to choose cells with fewer passages.
4. In the cell incubator (37 ℃, 5% CO₂) for 2-24 h.
Note: Too long culture time may cause some migrated cells to fall off from the bottom surface of the membrane. It is recommended to select the appropriate culture time through pre-experiment.
5. Carefully take out the small chamber, suck out the culture medium in the upper chamber, and gently wipe off the unmigrated cells in the upper chamber with the end of a wet cotton swab.
Note: Wipe off the unmigrated cells in the upper chamber. The action must be gentle and do not puncture the polycarbonate membrane.
6. Transfer that chamber to a well containing 600 uL of Fixation Solution, fix at room temperature for 10 min, and wash 3 times with PBS.
7. Then transfer the chamber to 600L Staining Solution, stain at room temperature for 5-15 minutes, wash 3 times with PBS, 3 minutes each time, and 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.
8. Place the chamber in a clean hole, select at least 3 visual fields with the microscope to observe and take photos, and calculate the number of migrating cells.
9. 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 elution at a room temperature of 22-28C. 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.

Own instruments, reagents, consumables:
Migratory 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℃, 10 ℃), 5% CO₂
PBS
, cotton swabs, forceps, 96-well plate, 24-well plate, adjustable pipette gun and tip
microscope
microplate reader (can measure the absorbance at 570 nm)

Reagent preparation:
24-Well Migration Plate: read-to-use; Before use, balance to room temperature; Store at 4 ° C.
Fixation Solution: read-to-use type; Before use, balance to room temperature; Store at -20 ° C.
Staining Solution: read-to-use; Before use, balance 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, it is important to conduct a pre-experiment to determine the best experimental conditions for cell migration, such as cell number, cell culture time, and staining time.
1, in an ultra-clean workbench, the 24-well Migration Plate was equilibrated at room temperature for 10 min.
2, cells were diluted to 0.5-5.0&times with serum-free medium; 10⁶cells /mL, cell suspension was prepared.
Note: It is recommended to starve the cells overnight before doing cell migration.
3. 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 in the upper and lower chambers must not produce bubbles, so that the migration effect will be weakened; (2) Cell status is critical, try to choose cells with fewer passages.
4. The cells were cultured in a cell incubator (37℃, 5% CO₂) for 2-24 hours.
Note: Too long culture time may cause some migrating cells to fall off from the bottom surface of the membrane, so it is recommended to choose the appropriate culture time by pre-experiment.
5, carefully remove the chamber, suck out the medium in the upper chamber, and gently wipe off the non-migrated cells in the upper chamber with the end of a wet cotton swab.
Note: Wipe off the upper chamber non-migrated cells, the action must be gentle, do not Pierce the polycarbonate membrane.
6, the chamber was transferred to a well containing 600 uL Fixation Solution, fixed at room temperature for 10 min, and washed 3 times with PBS.
7, the chamber was then transferred to the well containing 600L Staining Solution, stained at room temperature for 5-15 min, washed 3 times with PBS for 3 min each time, and allowed to dry. (If the chamber still has dark staining, it is recommended to gently clean the chamber in a beaker with PBS for 3 times and let it dry).
Note: Both ambient temperature and reagent temperature will affect the time required for staining, but a short staining time will only affect the depth of the color. If the color is light, heating or prolonging the staining time can be used to achieve the desired staining effect, and dyeing at 37℃ for 15 min can ensure sufficient staining.
8. The chamber was placed in a clean well, and at least 3 fields of view were selected for observation and photo taking under the microscope, and the number of migrating cells was calculated.
9, after taking photos, add 400 &micro to the lower chamber; L Elution Solution, elution for 10 min, the Staining Solution was completely eluted, and the eluted solution was absorbed 200   µ L into a 96-well plate and OD was measured at 570 nm.
Note: (1) Usually in 22-28C room temperature environment, it takes 10 min to complete the elution, the elution time can be adjusted according to the temperature difference. (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.