Cell Invasion Assay Service
First Edition (Revised on April, 2016)
The purpose of this experiment is to detect cell invasion ability. Invasion experiment mostly detects the ability of tumor cell invasion. Add cells to be studied into the upper chamber of Transwell, and add FBS solution with a high concentration into the lower chamber. The cells will penetrate matrix glue and polycarbonate membranes under the influence of FBS solution. Record the number of transmembrane cells. The number of the cells in different treatment groups are different, which shows different invasion ability.
Period: 1~2 weeks
(1) Precool: Precool tips, EP tube, 24-well culture plate and Transwell at 4 degrees for half an hour.
(2) Envelop basement membrane: The Matrigel gel melts into a liquid at 4 degrees overnight. Dilute it with serum free DMEM medium at the rate of one to nine. 40ul/well diluted glue on upper surface of the Transwell membrane. Then put it into rsbiotech with the condition of 5％CO2 and 37 degrees for two hours.
(3) Hydrate basement membrane: Add 50ul serum free DMEM medium into each chamber, then put it into rsbiotech with the condition of 5％CO2 and 37 degrees for an hour. Then suck and abandon the medium.
(4) Prepare single cell suspension: Digest cells after transfection with 0.25% of trypsin, then collects it and centrifuge it at 1000rpm for 5 minutes. Abandon the supernatant and resuspend cells with serum free DMEM medium. Count and adjust density to 1x10e6/ml.
(5) Inoculate cells: Add 200ul monocyte suspension into the upper chamber of Transwell, and add 600ul DMEM culture including 30% FBS into the lower. Each group was set up with three holes. Make sure no bubble between the lower medium and polycarbonate membranes in the small Chambers. Put it into rsbiotech with the condition of 5％CO2 and 37 degrees for 72 hours.
(6) Fix and dye: Take out the Transwell and absorb the medium of the upper chamber. Wash it three times with PBS buffer. Use sterile wet cotton swab to gently wipe off the uninvaded cells and Martrigel glue. Fix it with 95% alcohol for 30 minutes and dye it with Crystal violet for 10minutes. Wash it more than 3 times and then dry in the air.
(7) Take photos and count: Examined it under a 20 times optical microscope. Count and calculate the average amount. And the experimental data were repeated three times.
【Provided by customer】
1. Cell lines for assay or disposing (Our company can provide common used cell lines).
2. Drugs and experiment scheme for cell experiment.
1. Providing the experimental data and images.
2. Providing the test report.