cancer cell invaion assay protocol_LX【DOC精选】.doc

BD BioCoat? Matrigel? Invasion Chamber Protocol (Catalog No. 354480, 354481) Li Xu 05 27 11 base on BD BioCoat? Matrigel protocol 1. MATERIALS REQUIRED 1)12 inserts (Catalog # 354480) in each of two 24-well BD Falcon TC Companion Tissue Culture Plates, stored at -20°C in the original packaging. 2) Bicarbonate based culture medium such as DMEM (serum-free) 3) Control Inserts (24-well, Catalog #354578 or 6-well, Catalog #354576) 4) Chemoattractant such as 5% fetal bovine serum in tissue culture medium 5) Companion Plates (24-well, Catalog #353504 or 6-well, Catalog #353502) 6) Diff-Quik staining kit (Allegiance Catalog # B4132-1A) or other suitable fixative and stain 7) Scalpel (#11 blade recommended) 8) Microscope slides and coverslips 9) Cotton swab 10) Sterile forceps 11) Immersion oil INATRUMENTS REUQIRED 1) Microscope with (optional) camera 2) Humidified tissue culture incubator, 37°C, 5% CO2 atmosphere 3) Laminar flow tissue culture hood 2. PROCEDURE 1.0 Rehydration 1.1 Remove the package from -20°C storage and allow coming to room temperature. 1.2 Add warm (37°C) bicarbonate based culture medium (SFM) to the interior of the inserts and bottom of wells. Allow to rehydrate for 2 hours in humidified tissue culture incubator, 37°C, 5% CO2 atmosphere. Change CM to SFM and starve cells for 2 h (or overnight) for better invasion result. 1.3 After rehydration, carefully remove the medium without disturbing the layer of Matrigel? Matrix on the membrane. 2.0 Invasion Studies 2.1 Rehydrate the number of Matrigel inserts to be used as directed above. Prepare an equal number of Control Inserts by using sterile forceps to transfer them to empty wells of the BD Falcon? TC Companion Plate. 2.2 Prepare cell suspensions in (serum-free) culture medium containing 5x104 cells/ml (0.5 ml each well, and 0.5x6 = 3 ml for each sample) for 24- well chambers or 1.25x105 cells/ml for 6-well chambers. (after trypsin digestion, wash cell 1-2 times using PBS, and then re