Buoyancy enabled separation method and system

A buoyancy enabled separation method for isolation from a sample including a variety of different cells a sparse subset of cells that is differentiated by a plurality of different cell surface markers, comprising:

• a) placing the sample in a container;
• b) adding first flexible shell microbubbles having a flexible shell with an inner bubble wall enclosing a gas and an outer bubble wall conjugated to a first antibody capable of binding to a first cell surface marker of a first subset of cells encompassed in the sample;
• c) incubating over a time span sufficient to allow an interaction between the first antibody and the first cell surface marker binding the first antibody to the first cell surface marker;
• d) allowing the first flexible shell microbubbles with the first subset of cells bound to the first flexible shell microbubbles to separate by flotation from a remainder of the sample;
• e) removing waste from the container, including cells within the sample other than the first subset of cells;
• f) disrupting the first flexible shell microbubbles such that the first set of isolated cells are no longer buoyant;
• g) adding second flexible shell microbubbles having a flexible shell with an inner bubble wall enclosing a gas and an outer bubble wall conjugated to a second antibody capable of binding to a second cell surface marker of a second subset of cells encompassed within the first subset of cells;
• h) incubating over a time span sufficient to allow an interaction between the second antibody and the second cell surface marker binding the second antibody to the at least one second cell surface marker;
• i) allowing the second flexible shell microbubbles with the second subset of cells bound to the second flexible shell microbubbles to separate by flotation from a remainder of the sample;
• and
• j) removing waste from the container, including cells within the first subset of cells other than the second subset of cells.