Protocol 7

7. Dynamic Flow Assay in a Parallel Plate Flow Chamber

John T. Patton~GlycoTech Corporation, Rockville, Maryland 20850

Flow assays allow visualization of cell adhesion under well-defined wall shear stress. The visualization of the different events of cell adhesion can be quantified by selective image acquisition and subsequent image processing. Flow assays are uniquely suited to the investigation of adhesive events which occur very rapidly in a time scale shorter than that of most static adhesion assays. In addition, events subsequent to the initial events can be studied such as cell stabilization and spreading giving some insight into the kinetics of particular cell-cell or cell-substrate adhesive behavior.

Materials:

Microscopy:
Inverted-stage microscope configured for phase contrast and fluorescence operation

Objective lens (6.3 x, 10 x, 40 x)

Stage incubator
Biological:
Cell suspension (neutrophils, cancer cells)

Cell monolayer or coated substrate in 35 mm dishes

Adhesion media (culture media serum-free with 12 mM Hepes)

Fibronectin (human plasma)

Bovine serum albumin (BSA)

Flow Chamber System:
Flow chamber deck with gasket and tube fittings

35 mm tissue culture dishes

Harvard syringe pump

Glass syringes to fit on pump (3, 5, 20, or 60 cc)

Silastic tubing

Stopcocks

Vacuum pump
Computer/Electronics:
Digital image acquisition and processing system

Display monitor

Video recorder

CCD camera with controller

Time-date generator

Computer with interface to image system

Storage device for images

Protocol:

Cell Monolayer Preparation

1. Coat dishes with human fibronectin (FN) by adding 1 ml of 5.5µg/ml FN solution to each dish.

2. Incubate for 30 min. at room temperature.

3. Aspirate solution out of each dish.

4. Add 2 ml of cell suspension (Huvecs, CHO cells) to each dish at seeding density of 0.5-3.0 x 10⁵ cells/ml depending on time required to reach confluence.

5. After 1-5 days, use dishes with confluent monolayer in flow assay. Feed cells every 2-3 days, but usually not with 48 hrs. of flow assay.

Coated Substrate Preparation

1. Outline a coating region (5 mm diameter) for coating substrate in the center of each dish with marking pen.

2. Add 20µl of solution containing substrate at a concentration of 10µg/ml to coating region. Incubate 1 hr.

3. Aspirate off liquid, add 20µl of 1% BSA to block for 1 hr.

4. Aspirate off BSA, add 20µl of inhibitor for 1 hr.

5. Dishes are ready for use in flow assay.

Flow Assay Using Parallel Plate Flow Chamber

1. Turn on stage incubator and warm adhesion media to 37°C 1 hr. prior to beginning flow assay.

2. Assemble flow system apparatus connecting inlet, outlet, and vacuum lines to the flow chamber deck. Fill system with media and remove all air from system.

3. Fill inlet reservoir with cell suspension. For assays using cell monolayers, 10⁶ cells/ml is recommended. For coated substrate assays, use 10⁵ cells/ml.

4. Attach dish to flow chamber deck by holding the deck inverted, place a small bubble of media on flow path area, then place 35 mm dish on the deck. Vacuum will hold dish on deck. Make sure dish was attached with no air bubbles in the flow path.

5. Place assembled chamber on microscope stage.

6. Initiate flow of cells syringe pump connected to outlet flow chamber at a shear stress in the range of 0.1-4.0 dynes/cm².

7. Allow cells to flow for sufficient time to get an adequate number of cells interacting with the cell monolayer or coated surface. Generally 3-10 min. is used.

8. Begin image acquisition. Collect images at 7-10 locations on the dish. Generally 3 dishes at a given experimental condition gives enough data to show statistical differences between treatments.

9. After images are acquired on all dishes, perform image analysis to quantify the flow assay.

References:

(1) Lawrence, M.B., McIntire, L.V., Eskin, S.K., (1987), Effect of flow on polymorphonuclear leukocyte/ endothelial cell adhesion, Blood 70: 1284-1290.

(2) Patton, J.T., Menter, D.G., Benson, D.M., Nicolson, G.L., McIntire, L.V., (1993), Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time, Cell Motility and the Cytoskeleton 26: 88-98.

(3) Jones, D.A., Abbassi, O., McIntire, L.V., McEver, R.P., Smith, C.W., (1993), P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells, Biophysical Journal 65: 1560-1569.