Figure 6.

Western blot analysis of captured PBMC extracts using anti-GAPDH, -α-tubulin, -FCN1, -CD14, -CD11a, -hemoglobin and -Erk1/2 antibodies. (A) LeukoCatch was equipped with various numbers of filters. Analyzed samples were 10-μL extracts (from 40 μL of blood) of PBMCs captured with the indicated numbers of filters in the LeukoCatch. In the 5 × 5 sample, the blood sample was passed 5 times through a 5-filtered LeukoCatch. Horizontal arrow or asterisk indicates the band for CD14 or immunoglobulin, respectively. (B) Passage frequency of the blood sample through the LeukoCatch was varied. Analyzed samples were 10-μL extracts of cells captured from the indicated number of passages through the LeukoCatch filters. Lanes 1-5 reflect blood washed twice with PBS (see steps e to h in Figure 1B). Lane 6 (sample 3-5) reflects blood washed 5 times with PBS. In lanes 7 or 8, a PBMC extract (2 μL from 160 μL blood or 10 μL from 0.8 mL blood) prepared by the standard method for PBMC preparation using centrifugation in the presence of Ficoll [11] was analyzed. As for CD11a, 100 μg extract of Jurkat cells was also examined using the same anti-CD11a antibody to confirm the identity of detected bands. (C) The number of PBS washes was varied. Analyzed samples were 10-μL extracts of cells captured by the LeukoCatch after the indicated number of PBS washes. (D) Western blots for estimation of the removed red blood cells from the filters by monitoring the captured hemoglobin. The leukocyte extract obtained by the serial two-fold dilution of non-treated blood samples (lanes 1-6) or the leukocyte extract using the 5-filtered LeukoCatch (lanes 7-11) were subjected to western blot and probed with anti-hemoglobin antibody. The bar graph in the lower panel shows the intensity of each band measured by using the Image-J software. Numbers above the bars represent the mean values of band intensities from three independent films of distinct exposure time of the western blots (1 min, 10 sec and 1 sec). Pairs of arrows or arrowheads represents the compared bands (see text).