Carbohydrate-Specific Adhesion of IntactCells 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.
|Acrylic adhesion chamber |
Acrylic transfer dish Glass-backed silica HPTLC plates
Buffered cell medium
Bovine serum albumin (BSA)
Phosphate buffered saline
Radiolabeled (or otherwise tagged) cell suspension on ice
150mm Petri dishes
X-ray film (or alternate cell detection system)
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�, 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 �/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 ch
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