A microfluidic device comprising an enclosure comprising: a support structure, a microfluidic circuit structure and a cover defining a microfluidic circuit; said microfluidic device further comprising a first electrode, an electrode activation substrate, and a second electrode, wherein said first electrode is part of a first wall of said enclosure and said electrode activation substrate and said second electrode is part of a second wall of said enclosure; wherein said microfluidic circuit comprises: a flow region configured to contain a flow of a first fluidic medium; one or more inlets through which said first medium can be input into said flow region; one or more outlets through which said first medium can be removed; a microfluidic channel comprising at least a portion of said flow region; and a microfluidic sequestration pen (436, 438, 440) comprising: an isolation structure comprising an isolation region configured to contain a second fluidic medium; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel and a distal opening into said isolation region, and a length L con of said connection region from said proximal opening to said distal opening is at least 1.0 times the width W con of said proximal opening of said connection region, wherein the microfluidic circuit is configured to permit mixing of components of said second medium with said first medium by diffusion or mixing of components of said first medium with said second medium by diffusion, while permitting no flow of said first medium from said flow region into said isolation region; and wherein said width W con of said connection region at the proximal opening is parallel to a direction of said flow of said first medium in said flow region. a support structure, a microfluidic circuit structure and a cover defining a microfluidic circuit; said microfluidic device further comprising a first electrode, an electrode activation substrate, and a second electrode, wherein said first electrode is part of a first wall of said enclosure and said electrode activation substrate and said second electrode is part of a second wall of said enclosure; wherein said microfluidic circuit comprises: a flow region configured to contain a flow of a first fluidic medium; one or more inlets through which said first medium can be input into said flow region; one or more outlets through which said first medium can be removed; a microfluidic channel comprising at least a portion of said flow region; and a microfluidic sequestration pen (436, 438, 440) comprising: an isolation structure comprising an isolation region configured to contain a second fluidic medium; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel and a distal opening into said isolation region, and a length L con of said connection region from said proximal opening to said distal opening is at least 1.0 times the width W con of said proximal opening of said connection region, a flow region configured to contain a flow of a first fluidic medium; one or more inlets through which said first medium can be input into said flow region; one or more outlets through which said first medium can be removed; a microfluidic channel comprising at least a portion of said flow region; and a microfluidic sequestration pen (436, 438, 440) comprising: an isolation structure comprising an isolation region configured to contain a second fluidic medium; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel and a distal opening into said isolation region, and a length L con of said connection region from said proximal opening to said distal opening is at least 1.0 times the width W con of said proximal opening of said connection region, an isolation structure comprising an isolation region configured to contain a second fluidic medium; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel and a distal opening into said isolation region, and a length L con of said connection region from said proximal opening to said distal opening is at least 1.0 times the width W con of said proximal opening of said connection region, wherein the microfluidic circuit is configured to permit mixing of components of said second medium with said first medium by diffusion or mixing of components of said first medium with said second medium by diffusion, while permitting no flow of said first medium from said flow region into said isolation region; and wherein said width W con of said connection region at the proximal opening is parallel to a direction of said flow of said first medium in said flow region.