Combined use of micropipette aspiration and perifusion for studying red blood cell volume regulation

We combined micropipette aspiration with a new technique of perifusion to study volume behavior of single red blood cells (RBCs) during anisotonic conditions. Other techniques have suggested that RBC volume changes are complete within 1 s, but our pipette data indicate a slower volume response of 5-10 s. This difference appears to be due to the partial aspiration of the cell into the pipette, which in part prevents exposure of the membrane area to the anisotonic medium. However, we found that medium will pass the aspirated RBC (-20 mm H2O aspiration pressure) and enter the pipette at a volume rate of 0.3 micron3/s; this rate was measured by introducing a flow marker, a separate RBC or a cell fragment, into the pipette. The osmotic balance across the RBC also affected the flow: if the outside fluid was made hypertonic, the pipette flow decreased, but a hypotonic conditions, the flow increased and contributed to the exposure of the aspirated portions of the RBC. However, after the initial 10 s, the RBC geometry can be precisely monitored; at 200 mOsm, the RBC swelled by 41.0 +/- 0.9% and then demonstrated a small but significant (P < 0.001) regulatory volume decrease. We suggest that this technique is very precise in measuring the dynamics of geometric regulation, although acute changes are affected by the partial cell aspiration and pipette flow.