Author(s): Okuzaki D, Kimura S, Yabuta N, Ohmine T, Nojima H
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Abstract BACKGROUND: Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. However, lack of a simple preparation technique has limited the use of such extracts. The aim of this study is to develop a simple and easy system that can selectively obtain leukocyte extracts without hemoglobin. METHODS: A filter that captures the leukocytes but not RBCs was set at the bottom of a 10-mL medical syringe by sandwiching it between plastic stoppers. The capturing efficiency of leukocytes with this tool, called LeukoCatch, was examined using human macrophage cells (MONO-MAC-6). The abilities of LeukoCatch system to capture the leukocyte proteins and to remove the hemoglobin from RBCs were tested by western blot analysis using human blood samples. RESULTS: This study presents the development of LeukoCatch, a novel tool that allows the preparation of leukocyte extracts from blood samples within 3 min without centrifugation. Tissue-cultured human macrophage cells were tested to determine the optimal filter numbers and pass-through frequencies of LeukoCatch, which was then applied to 2-mL blood samples. Samples were passed 2~5 times through a LeukoCatch equipped with 5 filters, washed twice with phosphate-buffered saline for red cell removal, and leukocyte proteins were extracted with 0.5 mL of elution buffer. Western blot analysis of the purified extract indicated that more than 90\% of hemoglobin was removed by the LeukoCatch and that the protein recovery rate of leukocytes was at least 4 times better than that of the conventional centrifugation method. CONCLUSION: We conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.
This article was published in BMC Clin Pathol
and referenced in Journal of Genetic Syndromes & Gene Therapy