Kinetic Analysis of Endocytosis by XWNj40k

VIEWS: 2 PAGES: 2

									   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.

								
To top