Effect of Hypoxia Exercise on Erythrocyte Aggregability

The total erythrocyte aggregation was determined by using Microfluidic Ektacytometer (Rheoscan-AnD 300, RheoMeditech, Seoul, Korea).

To assess cardiopulmonary capacity, the cardiopulmonary exercise test (CPET) on a cycle ergometer was performed 4 days before and after the intervention. All subjects underwent exercise with a mask to measured oxygen consumption (VO2) breath by breath using a computer-based system (Master Screen CPX, Cardinal-health Germany). After 5-min baseline resting period records, a 2-min warm-up period (60 rpm, unloaded pedaling) was started and followed by an incremental work (30 W elevation for each 3-minute) until exhaustion (i.e., progressive exercise to maximal O2 consumption, VO2max).

• The erythrocyte suspension (1×10^6 cells/μl) was incubated with fibrinogen (400 mM) and anti-fibrinogen-FITC monoclonal antibody (1:500, Cedarlane) in the dark for 30 min at RT.
• After the staining is completed, add 1μl suspension into 100μl medium solution, then rotate the sample on a Viscometer (Brookfield) at different rates (0, 50, 100, 200, 400, 800 rpm) for 1 min at 37°C in the dark.
• The mean fluorescence intensity (MFI) obtained from 50,000 erythrocytes was measured by FACSCalibur (Becton Dickinson, New Jersey, USA).

Erythrocyte suspensions (1x10^6 cells /µl) were incubated with monoclonal anti- CD147(eBioscience) and anti-CD47(BioLegend) that were conjugated with FITC in the dark for 30 min at 37°C. The MFI obtained from 50,000 erythrocytes was measured by using FACSCalibur.

Erythrocyte suspensions (1x10^6 cells /µl) were incubated with monoclonal anti-CD49d (eBioscience) and anti- CD36(BioLegend) that were conjugated with FITC in the dark for 30 min at 37°C. The MFI obtained from 50,000 erythrocytes was measured by using FACSCalibur.

• The hematocrit (Hct) of erythrocyte sample was adjusted to 40% to eliminate the effect of dehydration after exercise.
• The adjusted sample was pre-treated with 400 mM fibrinogen for 30 min.
• The erythrocyte aggregation was determined by using Microfluidic Ektacytometer (Rheoscan-AnD 300, RheoMeditech, Seoul, Korea).

• The hematocrit (Hct) of erythrocyte sample was adjusted to 40% to eliminate the effect of dehydration after exercise.
• The adjusted sample was pre-treated with 10 mM Dextran for 30 min.
• The erythrocyte aggregation was determined by using Microfluidic Ektacytometer (Rheoscan-AnD 300, RheoMeditech, Seoul, Korea).

• The hematocrit (Hct) of erythrocyte sample was adjusted to 40% to eliminate the effect of dehydration after exercise.
• For excluding the effect of changed plasma composition, the erythrocyte sample was added into the platelet-poor-plasma (PPP) collected before the hypoxia exercise test.
• The erythrocyte aggregation was determined by using Microfluidic Ektacytometer (Rheoscan-AnD 300, RheoMeditech, Seoul, Korea).