McClean: 96 Well Plate Assay

Reagents

• Nunc 96-well coverslip bottom 96-well plates (Nunc #265300; stored in microscopy room)
• concanavalin A (conA) solution (2mg/ml concanvalin A MP Biomedicals, cat no: 150710; 5mM CaCl2 5mM MnCl2, located in -20C in 1ml aliquots).

Procedure

1. Remove conA from the -20C and allow it to thaw. ConA can be left at room temperature for several days without losing it efficacy (particularly for 96-well plate experiments where the adhesion of the cells to the coverslip doesn’t need to be that strong)
2. For screening and pilot experiments, use cleaned and/or previously used glass-bottom plates. For “actual experiments” use a brand new plate.
3. Prepare the wells to be used by filling them with 30ul of concanavalin A and allowing the conA to incubate for 5 minutes (the incubation time is not crucial). Remove the conA by aspiration.
4. Cells should be diluted so that when 100ul of the cell culture is loaded in a well, they “sparsely” cover the bottom of the coverslip, even after they have been allowed to settle. The desired amount of cells will need to be determined empirically. You should also decide whether or not you want to sonicate your cells before microscopy. For Mat a FY haploids at Klett 40, a good starting point is a 1:20 dilution (5ul of cell culture in 95ul of media per well).