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Abstract
BackgroundWhole-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.MethodsA 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.ResultsThis 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.ConclusionWe conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.
Highlights
Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research
We surmised that the leukocytes but not RBCs of the peripheral blood would be efficiently captured by passing through a small number of layered filters (Figure 1B-i) by moving the piton up and down by hand, and that the hemoglobin and immunoglobulin would be removed by washing the filter with phosphate-buffered saline (PBS) (Figure 1B-ii) with upward and downward piton movement
Optimal filter number in the LeukoCatch to capture macrophage cells To determine the optimal number of filters in the LeukoCatch, we examined the capturing efficiency of human macrophage cells (MONO-MAC-6)
Summary
Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. Novel approaches or revolutionary tools for transcriptomics are rapidly emerging [6]. These data reveal the up- or down-regulated genes that can serve as potential RNA diagnostic markers of relevant diseases [7,8,9,10]. If the marker gene products (mostly proteins) could be detected by antibodies using the extracts of leukocyte subpopulations in the PBMCs, the transciptome data would be practically useful for bedside diagnosis. The instrumentation required for RNA diagnostics is not routinely available at the bedside or in a common clinical facility
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