Protocol 9

9. Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates

Ronald L. Schnaar~Professor, Johns Hopkins University Medical School, Baltimore, Maryland 21205

This procedure allows detection of specific cell adhesion to glycolipids resolved on TLC plates. The steps include: (1) HPTLC of glycoconjugates, (2) coating the chromatogram with polymer, (3) preblocking the chromatogram to reduce non-specific cell binding, (4) mounting the chromatogram in the acrylic chamber, (5) adding labeled cells, (6) incubating, (7) removal of non-adherent cells by centrifugation, and (8) fixation of adherent cells for detection. Adherent cells never pass through an air/liquid interface prior to fixation, allowing detection of specific cell adhesion even if adhesive strength is low.

Materials:

Acrylic adhesion chamber
Acrylic transfer dish Glass-backed silica HPTLC plates

Polyisobutylmethacrylate

Chloroform

Hexane

Buffered cell medium

Bovine serum albumin (BSA)

Phosphate buffered saline

Radiolabeled (or otherwise tagged) cell suspension on ice

150mm Petri dishes

Glutaraldehyde

X-ray film (or alternate cell detection system)

Protocol:

1. Prescored 10 x 10 cm HPTLC plates are broken along one score to generate a 5 cm x 10 cm plate. Glycolipids are applied and TLC's developed using standard procedures. Chromatograms are then dried thoroughly (50°C, 1 hr.) and allowed to cool prior to polymer coating.

2. Polyisobutylmethacrylate (see pg. 99) is prepared as a 10% (w/v) solution in chloroform which is diluted 1/100 into rapidly stirring hexane to generate a 1 mg/ml stock which can be stored at room temperature. The solution is further diluted immediately before use with additional hexane to a final concentration of 2-200 ľg/ml depending on the cell type. Developed and dried TLC plates are dipped sequentially for 30 sec. each in hexane, then in PIBM solution, then allowed to air dry.

3. The PIBM-coated plate is immersed in medium until wet, then transferred into medium containing blocking agent 0.5 mg/ml BSA) for 30 min., then into medium without blocker.

4. The preblocked plate is placed sorbent side down on the spacers of the adhesion chamber. Medium is pipetted onto the back of the TLC plate and the rubber gasket applied such that air bubbles are excluded from the plate-gasket contact area. The gasket and acrylic cover are fixed in place with the screws supplied.

5. Any medium trapped in the chamber is decanted through the luer lock openings at the end of the Chamber. Cell suspension (10⁵-10⁶ cells/ml) in medium containing 1-5 mg/ml BSA is pipetted into one of the openings until the chamber is full and free of air bubbles (chamber capacity 15 ml). The end openings are sealed with luer locks, and the sealed box is placed with TLC sorbent side up such that the cells settle onto the plate surface. The box is placed on a microplate centrifuge carrier and centrifuged at 30xg, for 3 min. to bring cells into contact with the TLC plate surface.

6. The chamber is immersed (sorbent side up) in a water bath typically at 37°C for 30 min. to allow adhesion to occur.

7. The chamber is removed from the water bath, inverted, placed (sorbent side down) in a centrifuge carrier, and centrifuged (250-500xg, 10 min., 4°C to remove non-adherent cells from the TLC plate surface.

8. While still inverted, the chamber is fully immersed in an ice-cold tub of PBS, the cover and gasket removed, and the TLC plate is gently removed, righted (while immersed) and placed in the transfer dish. The fluid-filled dish is then used to transfer the TLC plate through two 150 mm Petri dishes filled with PBS to wash.

9. Still in the transfer dish, the plate is placed in an empty 150 mm Petri dish and gently overlaid with 100-150 ml of 2% glutaraldehyde in PBS at ambient temperature for 30 min.

10. The TLC plate is removed from the transfer dish, washed by immersion in PBS and placed vertically to dry.

11. The thoroughly dried plate is placed on X-ray film and exposed. After exposure for 1-24 hr. the film is processed to detect the position of adherent cells.

Note: If glycolipids are present at sufficient concentration (>500 pmol/lane) they may be detected on the TLC plates after autoradiography by incubating the plate in Coomassie Blue (0.3mg/ml) in methanol: water (1:5) for 30 min. followed by destaining in the same solvent mixture for 5 min. Coomassie Blue treatment also stains adherent cells which can be visualized by light-field microscopy at the sites of autoradiographic bands to confirm that the radiolabel detected corresponds to intact cell adhesion.

References:

(1) Swank-Hill, P., Needham, L.K., and Schnaar, R.L. (1987) Carbohydrate-specific cell adhesion directly to glycosphingolipids separated on thin-layer chromatography plates. Analytical Biochemistry, 163: 27-35

(2) Stroud, M.R., Handa, K., Ito, K., Saylan, M.E.K., Fang, H., Levery, S.B., Hakomori, S., Reinhold, B.B., and V.N.Reinhold (1995) Myeloglycan, a series of E-selectin-binding polylactosaminolipids found in normal human leukocytes and myelocytic leukemin HL-60 cells. Biochemical and Biophysical Research Communications, 209: 777-787.