Cell Characterization Protocols

(These protocols utilize Florescence Activated Cell sorting (FACs)) 

1) Check cells under microscope to make they are healthy before proceeding.

2) For optimal results, the cells should be in the mid to late log phase of growth (at or near confluence).

3) Aspirate old media, wash with PBS, and aspirate.

4) Add 3-4 mL of trypsin to cells and incubate 3-5 minutes. Cells should appear rounded and will begin to come

    up off the dish.

5) Triturate trypsin to get cells into suspension. Check flask under microscope to be sure that all cells are in

    suspension.  If cells are still attached wait an additional 5 minutes and triturate again. If cells continue to adhere,

    add an additional 3-4 mL of trypsin, wait 5 minutes, and repeat trituration step.

6) Add media (use volume equal to volume of trypsin used) containing 20% FBS to deactivate trypsin, triturate

    thoroughly.

7) Strain cells through 40 um filter into 50 mL conical tube to remove clumps.

8) Centrifuge at 2500 x g x 5 minutes, resuspend in 10 mL PBS - Count cell density with Coulter Counter

For Lectin binding experiment - transfer 1x10^6 cells in 1 mL PBS to 5.0 mL sterile tubes.

LECTIN PROTOCOL

Tube 1 - Cell suspension only

Tube 2 - 1 mL cells + 5 uL Helix pomatia - mix gently.

Tube 3 - 1 mL cells + 5 uL Griffonia simplicifolia - mix gently.

Incubate at room temp for 15 minutes, take to FACS for evaluation.

ANTIBODY PROTOCOLS

PECAM -1

Place 1 mL cells in 2mL microfuge tube - centrifuge at 2500 x g x 5 minutes. Aspirate PBS.                   

Place 50 uL Fixative A (Fix and Perm kit) onto cells, triturate, let sit for 15 minutes.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate PBS and resuspend cells in 1 mL PBS.

Add 50 uL PECAM-1 to cells + 50 uL Fixative B, mix gently, let sit for 30 minutes.

Add 250 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate, and resuspend cells in 1 mL PBS.

Add secondary antibody at 1:100, let sit for 30 minutes in dark.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate, resuspend cells in 1 mL PBS.

Place in FACs tube for evaluation.

           

vWF -

Place 1 mL cells in 2 mL microfuge tube - centrifuge at 2500 x g x 5 minutes. Aspirate PBS.                   

Place 50 uL Fixative A (Fix and Perm kit) onto cells, triturate, let sit for 15 minutes.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate PBS and resuspend cells in 1 mL PBS.

Add 50 uL vWF to cells + 50 uL Fixative B, mix gently, let sit for 30 minutes.

Add 250 uL PBS, centrifuge 5 minutes. Aspirate, and resuspend cells in 1 mL PBS.

Add secondary antibody at 1:100, let sit for 30 minutes in dark.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate, resuspend cells in 1 mL PBS.

Place in FACs tube for evaluation.

VE-Cadherin -

Place 1 mL cells in 2mL microfuge tube - centrifuge at 2500 x g x 5 minutes. Aspirate PBS.                   

Place 50 uL Fixative A (Fix and Perm kit) onto cells, triturate, let sit for 15 minutes.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate PBS and resuspend cells in 1 mL PBS.

Add 50 uL VE-Cadherin to cells + 50 uL Fixative B, mix gently, let sit for 30 minutes.

Add 250 uL PBS, centrifuge 5 minutes. Aspirate, and resuspend cells in 1 mL PBS.

Add secondary antibody at 1:100, let sit for 30 minutes in dark.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate, resuspend cells in 1 mL PBS.

Place in FACs tube for evaluation.

eNOS -

Place 1 mL cells in 2 mL microfuge tube - centrifuge at 2500 x g x 5 minutes. Aspirate PBS.                       

Place 50 uL Fixative A+ 50uL Fixative B (Fix and Perm kit) onto cells, triturate, let sit for 15 minutes.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate PBS.

Resuspend cells with 1 mL PBS.

Add 50 uL eNOS to cells, mix gently, let sit for 60 minutes.

Add 500 uL PBS, centrifuge 5 minutes, aspirate, resuspend with 1 mL PBS.

Place in FACs tube for evaluation.

alpha smooth muscle actin - 

Place 1 mL cells in 2 mL microfuge tube - centrifuge at 2500 x g x 5 minutes. Aspirate PBS.             

Place 50 uL Fixative A+  50 uL Fixative B (Fix and Perm kit) onto cells, triturate, let sit for 15 minutes.

Add 500 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate PBS.

Aspirate, resuspend with 1 mL PBS.

Add 50 uL VE-Cadherin to cells, mix gently, let sit for 30 minutes.

Add 250 uL PBS, centrifuge at 2500 x g x 5 minutes. Aspirate PBS.

Resuspend cells with 1 mL PBS.

Add secondary antibody at 1:100, let sit for 30 minutes in dark.

Add 500 uL PBS, centrifuge 2500 x g x 5 minutes. Aspirate PBS. Resuspend cells with 1 mL PBS.

Place in FACs tube for evaluation.

Endothelial cells should be positive for all antibodies EXCEPT alpha smooth muscle actin.

                        Reagents                              Vendor                                           Catalog #

                        alpha smooth muscle actin       Abcam                                          32575

                        Donkey anti-sheep IgG            Jackson Immuno Research              713-095-147

                        eNOS3                                  Santa Cruz                                     sc-654

                        G. simplicifolia                        EY Labs                                        F-2401-2  

                        H. pomatia                             EY Labs                                        F-3601-1

                        Normal mouse IgG1                Santa Cruz                                    sc-2855

                        Normal mouse IgG1                Santa Cruz                                    sc-2866

                        Normal rabbit IgG                    Santa Cruz                                    sc-3870

                        PECAM-1                               Santa Cruz                                    sc-71873

                        VE-Cadherin                           Santa Cruz                                    sc-9989PE

                        vWF                                       Affinity Biologicals                         SARTW-IG

                      

                       Supplies

                       FACs  5mL tube                      Falcon                                          352063

                       Fix and Perm Kit                     Caltag                                           GAS-004