LeukoCatch, a quick and efficient tool for the preparation of leukocyte extracts from blood
© Okuzaki et al; licensee BioMed Central Ltd. 2011
Received: 6 May 2011
Accepted: 17 August 2011
Published: 17 August 2011
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.
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.
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.
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.
Keywordscell extract leukocyte diagnosis PBMC proteomics
Peripheral blood is an ideal surrogate tissue for disease diagnosis and prognosis. Extensive data have been accumulated from the genome-wide gene-expression profiling of patient peripheral blood mononuclear cells (PBMCs) and their leukocyte subpopulations using DNA microarray analyses [1, 2] in a wide range of diseases, including cancer [3, 4] and autoimmune diseases . Moreover, novel approaches or revolutionary tools for transcriptomics are rapidly emerging . These data reveal the up- or down-regulated genes that can serve as potential RNA diagnostic markers of relevant diseases [7–10]. 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. Generally, the instrumentation required for RNA diagnostics is not routinely available at the bedside or in a common clinical facility.
Most antibody-based diagnostics target proteins secreted into the serum or protruding from the cell membrane surface. Such targets are easily detected by specific antibodies via enzyme-linked immunosorbent assay (ELISA) or dot blot analysis using the supernatant or sediments of the blood sample. To efficiently analyze leukocyte protein extracts for expression profiling by specific antibodies, the white blood cells (WBCs) or leukocytes must be separated from the red blood cells (RBCs) that normally occupy about half of the total blood volume. Red blood cells contain abundant iron-rich hemoglobin, which can hamper the analysis of the comparatively small amount of leukocyte proteins.
The separation of RBCs from leukocytes has been primarily based on methodology established through the pioneering work of Bøyum . Separation media consisting of a mixture of Ficoll 400 (or Percoll) and an iodinated density gradient medium have been very widely used to purify human leukocytes in basic research and in routine diagnostic studies. Nevertheless, variable extract recovery rates and/or possible changes in the expression profiles during the several-hour separation procedure can disturb the data reproducibility. Moreover, the leukocyte separation procedure requires an expensive centrifugation facility that is not routinely available at the bedside. Thus, a quick, easy, inexpensive, and efficient method for the purification of leukocyte extracts is needed.
In the present study, we present LeukoCatch, a novel tool for the centrifugation-free purification of leukocyte extracts from whole blood. LeukoCatch consists of a layer of filters held at the bottom of a syringe, which captures leukocytes but not RBCs from a blood sample. Captured leukocytes are washed by phosphate-buffered saline (PBS) and the protein extract of leukocytes were collected as the flow-through fraction that was obtained by flushing the filter with extraction buffer. Western blot analyses revealed that the collected extracts lacked most RBC components and retained a sufficient amount of leukocyte-derived proteins. We propose that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research.
Structure of LeukoCatch and its usage
Optimal filter number in the LeukoCatch to capture macrophage cells
Western blot analysis was performed using antibodies for GAPDH and Erk1/2, which are expressed by MONO-MAC-6 cells, using a 5-μL extract (10% of total) of escaped cells. The amount of passed-through proteins was reduced relative to the number of filters used, as judged by direct observation (Figure 2B) or quantification of the band intensities by using the Image-J software (Figure 2C). Similar western analysis using a 5-μL extract (10% of total) of captured cells indicated that the LeukoCatch with > 3 filters was practically useful for capturing these proteins. The band intensities were saturated due to abundant captured proteins when 3 to 10 filters were employed, revealing the excellent capture efficiency of the LeukoCatch. Since passing the sample through 7 or 10 filters is practically difficult (due to the sticky movement of the syringe plunger), we used 5 filters (i.e., a "5-filtered LeukoCatch") for further experiments.
Optimal passage frequency through the LeukoCatch for macrophage cells
Effect of filter number and passage frequency on leukocyte capture from blood samples
Western blot analysis to assess the leukocyte capture efficiency
To assess the extent of protein capture by the 5-filtered LeukoCatch system for blood samples, we performed western blot analysis for the proteins retained on the filter (retentate) after PBS wash, and then eluted into the flow-through fraction (Figure 1b, step j). We used anti-GAPDH and -Erk1/2 antibodies to detect proteins that exist in both leukocytes and RBCs. We also used antibodies to detect α-tubulin (expressed predominantly in leukocytes but less abundantly in RBCs), hemoglobin (expressed exclusively in RBC) and Ficolin 1 (FCN1: expressed exclusively in leukocytes; 15). To examine if the leukocytes harboring the membrane proteins may stick to filter fiber surfaces differentially and retained by the filters, we also tested antibodies against leukocyte specific membrane proteins such as CD14 and CD11a. FCN1 is a soluble collagen-like protein that binds sugar structures or acetylated compounds present on microorganisms and initiates activation of the lectin complement pathway . CD14 is a component of the innate immune system that is either anchored into the leukocyte membrane or in a soluble form . CD11a is a component of the Beta2-integrin CD11a/CD18 (LFA-1) that is a leukocyte transmembrane protein .
As expected from the results shown in Figure 5A, the capture efficiency of these proteins was increased when the sample was passed through twice rather than once (Figure 6B, lanes 1, 2), but similar results were obtained for passage frequencies of 2~5 times (Figure 6B, lanes 2-5). Notably, after washing with PBS 5 times for a 3-times passages through 5-filtered LeukoCatch (denoted by 3/5), the intensities of the bands for all proteins were dramatically reduced (Figure 6B, lane 6). Hemoglobin apperared to be removed during these repeated passages (Figure 6B, lanes 3-6).
When the PBS wash frequency was 2~5 times, the intensity of the FCN1 and hemoglobin bands were drastically reduced after 3~5 PBS washes (Figure 6C). These results indicate that the best protocol for the practical use of the LeukoCatch system is as follows; repeated (5 x) blood passages through a 5-filtered LeukoCatch with two PBS washes.
We compared these results with those of an extract prepared according to the conventional centrifugation of a blood sample on Percoll. A 10-μL extract derived from 40 μL of blood using the LeukoCatch showed an FCN1 band of similar intensity as that of a 2-μL extract derived from 160 μL of blood using the centrifugation method (Figure 6B, lanes 7 and 8). This result suggests that the protein recovery of the 5-filtered LeukoCatch was at least 4 times better than that of the conventional centrifugation method.
We also compared the amount of captured hemoglobin in the LeukoCatch without filters (Figure 6D, lanes 1~6) and with five filters (Figure 6D, lanes 7~11). We found that the band intensities of 2-4 dilution (4.1, arrow) of the former was similar to 2-0 (= 1) dilution (3.6, arrowhead) of the latter. This indicates that about 2-4 or 1/16 dilution of hemoglobin was attained by the LeukoCatch, namely, more than 90% of hemoglobin was removed by the LeukoCatch. Comparison of the intensity of other bands (arrows or arrowheads) also supports this conclusion.
The present study reports a quick, easy, efficient, and centrifugation-free purification system of leukocyte extracts from PBMCs, using a novel tool containing layers of Pall Leukotrap filters (Figure 1). The Pall Leukotrap RBC inline filter system  permits the near-complete depletion of leukocytes from blood . This filter system has been successfully applied to the bedside preparation of leukocyte-depleted platelet concentrates from whole blood or to remove exogenous infectious prions and endogenous infectious agents from red cell concentrates [18, 19]. In the present study, the usefulness of this filter was extended to the preparation of whole cell extracts from tissue culture cells or leukocytes from blood samples.
We first developed a novel tool called LeukoCatch that consists of a layer of filters held at the bottom of a syringe to capture leukocytes but not RBCs from a blood sample (Figure 1). Using human macrophage (MONO-MAC-6) cells, we determined the optimal filter numbers (Figure 2) and passage frequencies (Figure 3) for the LeukoCatch system. The system was then applied to human blood. The most practical protocol involved 2~5 passages through the 5-filtered LeukoCatch (Figure 1) with two PBS washes, followed by protein extraction with EB (Figures 4, 5 and 6). Western blot analysis probed by a typical leukocyte protein FCN1 showed that the 5-filtered LeukoCatch showed at least 4 times better recovery rate of leukocytes than the sample prepared by conventional centrifugation of a blood sample on Percoll (Figure 6B).
Although the Leukocatch system removes 90% of hemoglobin (Figure 6D), it is still be a huge contaminant in these preparations, considering that hemoglobin in RBC is at a concentration of 5 mM and about half the volume of whole blood is RBCs. However, western blot analysis using typical leukocyte proteins displayed sharp bands without distortion of their shapes (Figure 6A-C). Without this system, western blot analysis using the whole blood would not be practical because hemoglobin represents the major part of the total protein and hampers the detection of other leukocyte proteins.
Notably, these leukocyte membrane proteins (CD14 and CD11a) were also recovered into the collected samples, suggesting that even a population of leukocytes carrying membrane proteins is not retained by the filters. To increase the efficiency of present 30-40% (Figure 4 and 5), we can increase the number of stacked filters or increase the volume of syringe. However, this is practically unnecessary because the present system already can provide the enough amount of leukocyte extracts for 50 times western blots for typical leukocyte proteins FCN1, CD14 and CD11a from 2 mL blood sample, considering the results shown in Figure 6B obtained by using 40 μL of blood samples (40 μL × 50 = 2mL).
Since the LeukoCatch system is simple, an automatic machine may be easily developed for the large-scale preparation of protein extracts from blood samples. Elution of the trapped lymphocytes by flushing EB into the Leukotrap filters (which are commonly discarded after bedside use) may yield useful lymphocyte extracts for basic and clinical research. Since functional human lymphocytes can be recovered from Leukotrap filters by back-flushing with cold PBS , LeukoCatch could also be useful as a quick and easy tool for the recovery of live lymphocytes.
Taken together, 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. Notably, the protein recovery of the LeukoCatch was at least 4 times better than that of the conventional centrifugation method, which indicates the advantage of this technique.
Analysis with MONO-MAC-6 cells
MONO-MAC-6 cells (DSMZ, Germany) were maintained in RPMI1640 medium supplemented with 20% fetal bovine serum (FBS, HyClone, Logan, UT), 10 μg/mL human insulin, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin in a CO2 (5%) incubator at 37°C. Cells in the logarithmic growth phase were used for experiments.
MONO-MAC-6 cells (2 × 106 cells/experiment) suspended in 5 mL of medium were passed through (sucked-and-poured) twice by the LeukoCatch, and then were divided into two tubes. One tube was subjected to FACS analysis. The other tube was centrifuged twice at 190 × g for 5 min each at 25°C, with the pellet washed with 2.5 mL PBS between centrifugations. After removing the supernatant, 25 μL of elution buffer (EB) [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% TritonX-100, 1 mM EDTA (pH 8.0), 0.1 mg/mL PMSF, 1 mM Aprotinin, 0.001 mg/mL Leupeptin, 0.001 mg/mL pepstatin A, 1 mM NaF, 1 mM Na3VO4, and 10 mM β-glycerophosphate] were added to resuspend the pellet. Finally, 12.5 μL each of H2O and 4× sample buffer (SB) [0.2 M Tris-HCl (pH 6.8), 8% SDS, 0.2% bromophenol blue, 20% glycerol, 10% beta-mercaptoethanol] were added. The final suspension was used for western blot analysis.
Fluorescence activated cell sorting (FACS)
MONO-MAC-6 cells (2 × 106 cells/experiment) filtered by the LeukoCatch were stained by the CycleTEST™ PLUS DNA Reagent Kit (BD Bioscience) according to the manufacturer's instructions. Samples were divided into four tubes. Cells were counted four times for each sample to obtain the mean value using a FACScalibur with CellQuest software. This experiment was independently repeated twice.
Western blot analysis and antibodies
For western blot analysis, the cell extracts were boiled, separated by 8% or 10% SDS-PAGE, and transferred to a polyvinylidine difluoride (PVDF) filter (Millipore, Bedford, MA). The filters were incubated with rabbit polyclonal antibodies specific for ERK1/2 (Cell Signaling Technology, Danvers, MA), GAPDH (Fitzgerald Industries International, Concord, MA), hemoglobin (MBL, Nagoya, Japan) FCN1 (Atlas Antibodies AB, Sweden), CD14 (Abcam, Cambridge, UK) or CD11a (Abcam). Then, the filter was incubated with horseradish peroxidase-conjugated anti-rat IgG (Amersham, Piscataway, NJ). Immunoreactive protein bands were visualized using Renaissance™ chemiluminescence reagents (DuPont NEN, Boston, MA).
Application of LeukoCatch to blood samples
Blood samples (2 mL per tube) drawn from one of the authors (H.N, male, age 58) into EDTA vacuum blood collection tubes were sucked into the LeukoCatch syringe and poured back into the tube. This process was repeated for various numbers of times. Phosphate-buffered saline (5 mL) in a fresh container was sucked into the LeukoCatch syringe and poured back into the container. This process was repeated various times. Elution buffer (0.5 mL) was sucked into the LeukoCatch syringe and the dissolved protein extract was poured back to the tube. The protein extract in the flow-through fraction was either kept frozen at -80°C after addition of glycerol (20% final) for long-term storage, or was stored at -20°C after addition of 4× SB for western blot analysis.
Hematology analysis of blood samples
The number of RBCs, platelets (PLTs), and WBCs in the fresh blood samples treated with or without LeukoCatch immediately after the blood was drawn were counted with an LH 750 hematology analyzer (Beckman Coulter, Fullerton, CA). Triplicate samples were counted.
Preparation of PBMC extract by standard method
Fresh venous blood (2-4 mL) in EDTA (as an anticoagulant) was mixed with an equal volume of 2% dextran/saline solution and incubated at room temperature for 30 min to precipitate the RBCs. PBMCs in the supernatant were purified by density-gradient centrifugation on Percoll (density: 1.064 g/mL), and were subsequently suspended in SB for western blot analysis.
All of the experiments with use of samples of human blood were performed with the approval of the ethics committee of Osaka University.
List of abbreviations
- FACS :
fluorescence activated cell sorting
- GAPDH :
- LY :
- MO :
- NE :
- PBMC :
peripheral blood mononuclear cells
- PBS :
- PVDF :
- PTL :
- RBC :
red blood cell
- SB :
- WBC :
white blood cell
We thank Ms. Kana Ao of our laboratory for technical assistance and Mr. Shin Moto of Fukae Kasei Co., Ltd. for technical advice. We also thank Dr. Kate Edmondson and Dr. Patrick Hughes of Bioedit Ltd. for critical reading of the manuscript. This work was supported in part by Innovation Plaza Osaka of the Japan Science and Technology Agency (JST), and by grants-in-aid for Scientific Research on Priority Areas "Applied Genomics," Scientific Research (S), Exploratory Research, and the Science and Technology Incubation Program in Advanced Regions, from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Hiroshi Nojima. Hiroyuki Shimizu is a Research Fellow of the Japan Society for the Promotion of Science.
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