Formation of dense SS cells by in vitro O-D cycling. SS ND (1.080 to 1.090 g/mL) cells were isolated from SS whole blood by density gradient centrifugation using continuous density isotonic Percoll-Larex (Stractan) gradients with physiologic concentrations of Na⁺, K⁺, Mg²⁺, and Cl⁻. The ND cells were subject to O-D cycling in sealed flasks containing a bicarbonate-free HEPES buffer at pH 7.4 using 5% CO₂/balance air (duration, 5 minutes) and 5% CO₂/balance N₂ (duration, 10 minutes) for the oxy (O) and deoxy (D) stages, respectively. Buffer pH was adjusted to 7.4 after equilibration with 5% CO₂. Without adjustment, in the absence of bicarbonate and after equilibration with 5% CO₂, the resulting buffer pH was 6.8. Experiments using continuous deoxygenation or continuous oxygenation also used 5% CO₂-containing gasses. After O-D cycling (or continuous air or N₂ exposure) the flasks were flushed under sealed conditions with 100% oxygen for 30 minutes, followed by collection of the cells and a second density separation, as described above. Color transparencies of analytical gradients were taken and scanned by laser densitometry to quantitate the percentage of cells in each density fraction. Cell density fractions were defined as follows: LD (<1.080 g/mL), ND (1.080 to 1.090 g/mL), ID (1.090 to 1.114 g/mL), and HD (>1.114 g/mL). Results shown are representative of from 2 to 15 experiments and are paired results using a single SS patient in all arms of the experiment. (A) Effect of pH on dense cells formed after 22 hours of O-D cycling. (B) Time course of dense cells formed by continuous deoxygenation (5% CO₂/balance N₂) or O-D cycling at pH 6.8. (C) Dense cells formed after 22 hours of continuous oxygenation (5% CO₂/balance air) or O-D cycling at pH 6.8. (D) Effect of ATP depletion on dense cell formation by O-D cycling. The O-D cycling buffer contained either 10 mmol/L glucose (Gluc) or 10 mmol/L 2-deoxyglucose (D-Gluc). The final concentration of Ca²⁺ in the buffer was 1.5 mmol/L (for A and D) or 0.5 mmol/L (for B and C). B, density marker beads (Amersham Pharmacia Biotech, Piscataway, NJ).