How do I know ConA bead binding was successful?

After ConA bead binding, save an aliquot of supernatant [unbound fraction] and take an aliquot of sample slurry [bead fraction] for Trypan blue staining as described. Successful ConA bead binding is indicated when:

• The unbound fraction contains few cells/nuclei (Figure A).
• The bead fraction contains permeabilized, Trypan Blue positive cells surrounded by beads (Figure B).

Should you not observe this, ensure:

• Count/integrity of cells before binding to ConA beads.
• ConA beads were never frozen.
• Cells/nuclei were not clumped.
• Beads did not become clumped or dried out.
• All buffers were correctly prepared.

Figure. Unbound fraction from ConA bead prep shows few cells/nuclei (A), whereas the bead fraction contains permeabilized (Trypan Blue positive) cells/nuclei surrounded by beads (brown spheres).