ABSTRACT
【Objective】 To optimize the bottom and top(B&T) method for nucleated cell separation in cord blood in order to reduce the disturbance and improve the nucleated cell yield. 【Methods】 A new device comprised of two plastic plates and connection units was developed. B&T Cord blood bag was put between the plates, which were then centrifuged and put into the serum expresser together to go through serum and red blood cells separation simultaneously. After centrifugation parameter adjustment, the new Plates-B&T method was compared with traditional method in parallel. 【Results】 New Plates-B&T method could avoid buffy coat movement and disturbance. Compared to conventional PRP method, Plates-B&T method reduced centrifugation and separation time from(29.12±2.79)min to(14.91±2.12)min(P<0.05). Using new method, the recovery rate of total nucleated cell(TNC) was (89.04±5.19)%, the removal rate of red blood cell(RBC) was(55.56±22.57) %, and hematocrit(HCT) in buffy coat was(0.48±0.09)L/L. RBC removal was enhanced significantly, especially for cord blood samples with medium-low volume. 【Conclusion】 Based on B&T method, we established a new device and method, which increased the efficiency and quality of cord blood nucleated cell separation.
ABSTRACT
The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.