General protocol. Tumor cells (Fig. 1: 1) are incubated with biotin-tagged antibody. (Fig. 1: 2) Next, a suspension of cells is drawn through the channel. Wherever a CTC makes contact with a post, the biotin on its surface reacts with streptavidin, thus immobilizing labeled cells. (Fig. 1: 3) Next, the captured cells are fluorescently stained (Fig. 1: 4) and counted using fluorescent microscopy (Fig. 1: 5). Modifications to this procedure allow for measurement of the effects of anti-clumping reagent (Fig. 1: 6) and bystander white blood cells (Fig. 1: 7), as done in Part I. An additional pre-labeling step allows for tumor cells incubated under multiple antibody conditions to be run simultaneously in one channel at a particular flow rate, as done in Part II (Fig. 1: 8). Cell lines, antibody preparation and microchannel set-up are identical for all experiments run.
Schematic of capture zones. (a) Close up of the channel.
Capture efficiency vs. antigen density.
Streamlines around post.
k values vs. channel segment at various flow rates, plotted for four different effective antigen densities: (a) 60 000, (b) 30 000, (c) 15 000, and (d) 6000.
k values vs. flow rate of various channel segments, plotted for four different effective antigen densities: (a) 60 000 (b) 30 000 (c) 15 000, and (d) 6000.
Impact of flow rates upon percent of cells captured in: (a) K562 cells, (b) SKOV cells, and (c) T24cells.
Effect of high flow rate on percentage of SKOV and SKBR cells captured.
k value vs. effective antigen density of various channel segments, (a) with and (b) without bystander cells.
Percent of cells captured as a function of bystander cell concentration.
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