Micropipette

Red blood cell suspensions were adjusted to 20% Hct and incubated with varying concentrations of GBT440 or DMSO for 30 min in PBS containing 0.5% BSA. Red blood cells were then diluted to 0.5% Hct and deoxygenated by direct exposure to low oxygen gas, as a thin film of blood on an inclined glass plate was exposed to air in a cylindrical container (Fig. 1B). The equilibrated RBC suspension was transferred to a glass chamber attached to an isothermal stage regulated to 37°C (Sensortec, Inc.) and low oxygen gas was continually passed on top of the samples to prevent re-oxygenation. micropipettes (A.M. Systems) were pulled (P-97, Sutter Instruments) to an internal diameter of ~2 micron and opening angle of ~9°. The micropipette was attached on a pneumatic micromanipulator (Narishige, Japan) and secured to an inverted microscope (Olympus IMT-2, Tokyo, Japan). Aspiration pressure was induced hydrostatically and kept constant. Measurements were done with a high magnification 100X LUMPFL (Olympus, Tokyo, Japan) objective and a video system (Cohu, Inc. San Diego, CA). Subsequent aspiration length was measured off line from recorded images. Cell membrane elastic module and cell volume were calculated based on the relationship between pressure and aspiration length as previously reported [21 (link)].