Can I use two biotinylated antibodies for two positive isolations using the Anti-Biotin MultiSort Kit? 

We recommend to use two different indirect MACS MicroBeads for this approach. When performing multi-parameter sorting using the Anti-Biotin MultiSort MicroBeads for biotinylated primary antibodies, it is not possible to use a biotin-labeled antibody for the second separation as well. Since the Anti-Biotin MultiSort MicroBeads do not necessarily cover all biotin residues, it is impossible to ensure that the MicroBeads will only recognize the second biotin-conjugated antibody. For the second separation fluorochrome-coupled antibodies can be used in combination with Anti-Fluorochrome MicroBeads (e.g Anti-PE MicroBeads).