Flow Cytometric Analysis of Cell Surface LRP on U87 Cells Solution 1. Cell Dissociated Solution: Sigma Catalog# C-5789. 2. PBS/FBS: PBSa containing 1.5% heat inactivated fetal bovine serum and 0.1% sodium azide. 3. Mouse anti-human LRP monoclonal antibody 8G1 (Dr. Strickland DK, American Red Cross): diluted to 30 g/ml in PBS/FBS. 4. Goat anti-mouse Ig FITC: Becton Dickson Immunocytometry System, Cat. No. 349031, 1:12.5 diluted in PBS/FBS, keep in dark. Procedure: 1. Split the 1.0 x106 U87 cells into T-25 culture flasks and culture O/N. 2. Remove the medium, rince the cells with 2 mls of PBSa. 3. Incubate the U87 cells with 3 mls of prewarmed Cell Dissociated Solution in incubator for 2~3 min. 4. Sharply tap the flask to detach the cells, add 3 mls of cold normal U87 cell medium, and use the pepet up and down the cell suspension ~8 times to make the single cell suspension. 5. Transfer the cell suspension to 15 ml disposable centrifuge tube, spin at 1000 rpm for 2 min. 6. Suspend the cell pellet into 150 l PBS/FBS, and label the cells with FITC as following. Control Test 50 l cell suspension 50 l cell suspension 50 l PBS/FBS 50 l 1stAb 8G1 solution Incubate in ice-water bath for 45 min, wash 2 times with cold PBS/FBS by adding 2 mls of cold PBS/FBS and spinning at 1000 rpm for 2 min. 50 l Goat anti-mouse Ig FITC 50 l Goat anti-mouse Ig FITC Incubate in ice-water bath for 45 min in dark, wash 2 times with cold PBS/FBS by adding 2 mls of cold PBS/FBS and spinning at 1000 rpm for 2 min. Suspend the cells in 0.5 ml of PBS/FBS, run the flow cytometry.
Pages to are hidden for
"Kinetic Analysis of Endocytosis"Please download to view full document