Colloidal silica--aluminum modified--PVP density gradient centrifugation: centrifuge tube wall cell adherence, aggregation, separation properties and comparison to BSA and Ficoll.

A method is described for the inexpensive and easy preparation of colloidal silica--aluminum modified--polyvinylpyrrolidone (CS-AM-PVP) density gradient centrifugation medium. Using density gradient centrifugation, several cell separation and biochemical characteristics were studied: centrifuge tube wall cell adherence, mouse spleen cell density distribution, rebanding properties, mitogen response and cell aggregation. Cell adherence to the centrifuge tube wall using density gradients of CS-AM-PVP was compared with density gradients of bovine serum albumin and Ficoll. Few cells adhered to the centrifuge tube wall when CS-AM-PVP was used as a gradient medium; whereas, significant cell adherence to the centrifuge tube cell wall occurred when bovine serum albumin or Ficoll was used as a gradient medium. The CS-AM-PVP gradient medium did not inhibit the response to mitogens of mouse spleen cells which had been separated into density subpopulations in a discontinuous CS-AM-PVP density gradient, caused a minimum amount of cell aggregation, and was found to be non-toxic.     

Characterization of functionally distinct lymphoid and myeloid cells from human blood and bone marrow. I. Separation by a buoyant density gradient technique.

A buoyant density gradient procedure for mammalian cell separation is described. Gradients of Ficoll and Hypaque in balanced salt solution were used in a zonal rotor to separate distinct leukocyte populations from human peripheral blood and bone marrow. This method possesses the advantages of uniform osmolarity, density determination by refractometry, ease of preparation of gradient solutions, large sample capacity, and low viscosity with consequent ease of sterilization. Granulocyte-monocyte colony-forming units (CFU-C) from blood and bone marrow banded as a homogeneous peak with a density of 1.056 g cm-3 on steep gradients. Recovery of CFU-C was 36% with a 7- to 9-fold enrichment on shallow gradients. Both fractionated and unfractionated peripheral blood cells were stimulated by phytohemagglutinin (PHA), Concanavalin A (Con A), and allogeneic lymphocytes. In contrast, cells from unfractionated bone marrow and cells from the light density fraction of marrow were inhibited by Con A and responded variably to PHA.     

A separation chamber to sort cells and cell organelles by weak physical forces. V. A sector-shaped chamber and its application to the separation of peripheral blood cells

A separation chamber of 90 ml effective volume is described, that incorporates a sector shape (to prevent wall sedimentation) and anti-vortex lamellae (to prevent swirling upon acceleration and deceleration) as well as a flow-diverter for undisturbed layering and fractionation. The chamber is run an ordinary table centrifuge and possesses a resolving power for equilibrium density centrifugation that seems unsurpassed. Peripheral human blood cells were separated in a Percoll gradient yielding in 1 single run the concurrent isolation of 99% pure monocytes, 99% pure lymphocytes and 99% pure granulocytes. Recoveries varied from 94% to 102%. By modifying the shape of the density gradient, either 99% of all monocytes collected were more than 92.5% pure and at the same time separation of small versus large lymphocytes was obtained, or substantial purification of neutrophils and eosinophils was achieved. The potential for simultaneous separation of monocytes, B, T cells and granulocytes was established. Good separations were obtained with buffy coats from 1 U of blood. The chamber also functioned well in the standard Böyum method for mononuclear cell preparation.In addition, a method for the rapid removal of erythrocytes (within 9 min) at unit gravity is described with nearly quantitative yield of white cells.     

Low-stress Microfluidic Density-gradient Centrifugation for Blood Cell Sorting

Density gradient centrifugation exploits density differences between different blood cells to accomplish separation of peripheral blood mononuclear cells (PBMCs) from polymorphonuclear (PNM) cells, and erythrocytes or red blood cells (RBCs). While density gradient centrifugation offers a label-free alternative avoiding the use of harsh lysis buffers for blood cell isolation, it is a time-consuming and labor-intensive process during which blood cells are subject to high-levels of centrifugal force that can artifactually activate cells. To provide a low-stress alternative to this elegant method, we miniaturized and automated this process using microfluidics to ensure continuous PBMCs isolation from whole blood while avoiding the exposure to high-levels of centrifugal stress in a simple flow-through format. Within this device, a density gradient is established by exploiting laminar flow within microfluidic channels to layer a thin stream of blood over a larger stream of Ficoll. Using this approach we demonstrate successful isolation of PBMCs from whole blood with preservation of monocytes and different lymphocyte subpopulations similar to that seen with conventional density gradient centrifugation. Evaluation of activation status of PBMCs isolated using this technique shows that our approach achieves minimal isolation process induced activation of cells in comparison to conventional lysis or density gradient centrifugation. This simple, automated microfluidic density gradient centrifugation technique can potentially serve as tool for rapid and activation-free technique for isolation of PBMCs from whole blood for point-of-care applications.     

A novel method for the self-collection of blood, "thenar lancing phlebotomy" and investigation of its accuracy

"Thenar lancing phlebotomy" is a novel method for the self-collection of blood using a special phlebotomy device for the thenar and a self-collection blood kit. The thenar is punctured with a special lancet, the vein is subjected to automatic avascularization at the wrist and at the same time, a small centrifuge tube, attached to the suction cylinder of the device, is applied to the puncture and the blood is collected by suction. The small centrifuge tube containing the whole blood is centrifuged with a portable centrifugal separator to obtain plasma. In comparison with conventional finger prick phlebotomy, there is less pain and sufficient blood may be obtained. To investigate the accuracy of the method, we collected blood from the antecubital vein of 140 subjects and thenar lancing phlebotomy was simultaneously carried out on the same 140 subjects. The results of many blood tests currently included in medical check-ups were almost identical in the blood samples of both groups, suggesting that this method can be utilized in medical check-ups using self-collected blood sampies.     

Comparison between different pre-treatment techniques for sperm recovery prior to intrauterine insemination, GIFT or IVF

An attempt was made to compare different pre-treatment techniques in 16 normal and proven fertile and 30 subnormal and hitherto infertile semen samples. The techniques used were (i) standard, (ii) layering, (iii) discontinuous Percoll density gradient and (iv) albumin columns. Percoll gradient was most effective in separating a high fraction of progressive motile spermatozoa (75 and 57% in normal and subnormal semen samples, respectively). The albumin columns, as well as the standard techniques, were equally effective in recovering 45 and 24% respectively of progressive motile spermatozoa in normal and subnormal semen samples. The layering method was the least effective of the four techniques (4% recovery in normal and subnormal semen samples). In cases of contamination with inflammatory cells, the standard and layering methods were significantly (P less than 0.001) more advantageous than isolations with Percoll gradient and albumin columns. The percentage of ideal forms of spermatozoa recovered from a normal semen sample was significantly higher with the standard (P less than 0.01), layering (P less than 0.05) and Percoll gradient (P less than 0.05) techniques. In subnormal samples, only the Percoll gradient gave a significantly (P less than 0.02) higher percentage of ideal forms, whereas the other techniques were less effective. The significance and practical use of the various pre-treatment techniques are discussed in relation to the characteristics of the pre-treatment semen sample.     

On the pressure dependence of sedimentation coefficients. An experimental test of a common correction formula

The measurement of the pressure dependence of sedimentation coefficients is discussed. A new experimental method is described where the pressure in the solution is varied by layering different amounts of solvent or inert liquid over a solution layer of constant thickness. In this way the relative variation in pressure will be known very accurately. The experimental pressure variation of s determined by this method agrees within a few percent with the pressure dependence calculated from the pressure variation of viscosity, density, and partial specific volume.     

Detection of bacteria in blood by centrifugation and filtration.

Culture of blood is the most frequent means of diagnosing bacteremia. However, conventional blood culturing methods are slow in isolating bacteria. We developed a method for isolation of bacteria by centrifugation and filtration. Fresh human whole blood was inoculated with facultatively anaerobic and aerobic microorganisms (3 to 172 microorganisms per 5 ml). Seeded blood was then mixed with Ficoll-Hypaque (density, 1.149 +/- 0.002 g/ml) and centrifuged (386 x g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micron membrane filter (Millipore). The filters were then placed on chocolate agar plates and incubated at 35 degrees C in a humidified atmosphere containing 5% CO2. For each bacterium tested, approximately 35 to 100% of the viable microorganisms were recovered when compared with control cultures (pour plates of seeded blood). All bacteria produced isolated colonies on filters after overnight incubation (18 h). This procedure may prove to be a more rapid method for isolating bacteria from clinical blood samples than the blood culture bottle technique.     

Quantitative analysis of urine sediment using newly designed centrifuge tubes

We quantified the formed elements of urine sediment using newly designed plastic centrifuge tubes with top and bottom openings and a 0.5 ml sized bottom ball (YZ tube). This design minimizes the adherence of formed elements that occurs on the glass surface of conventional tubes. The numbers of white blood cells (WBC) and red blood cells (RBC) using glass tubes did not differ from those observed using YZ tubes. However, the YZ tube method detected renal casts more frequently than the conventional glass tube method; the detection rate for renal casts in normal urine samples was 21.4% vs 2.9%, in samples from hospitalized patients it was 47.5% vs 10.2%, and from patients with kidney disease it was 88.9% vs. 44.4%. Especially, the YZ tube method detected more hyaline casts in all types of samples. The correlation between the glass tube and YZ tube methods was good for WBC (r=0.996), RBC (r=0.964), and epithelial cell count (r=0.939), but the correlation was weak for casts (r=0.511 for hyaline casts; r=0.359 for other casts). In conclusion, the YZ tube method of urine sediment analyses is an easy and accurate quantitative method; it is recommended as the method of choice for detecting and quantifying pathological casts in urine. (received     

小鼠骨髓间充质干细胞的分离与培养

探索在体外分离培养小鼠骨髓间充质干细胞 (Bone m arrow m esenchym al stem cells,BMSCs)的最适条件。以密度梯度离心技术及贴壁筛选法相结合 ,在体外分离 BAL B/C小鼠的 BMSCs,通过 MTT法测定了不同离心力、不同换液时间、不同血清浓度、不同种植密度等因素对 BMSCs分离和培养的影响。在 5 0 0 g× 30 min的离心力下分离细胞 ,加入 10 %的胎牛血清 ,原代按 (12～ 2 0 )× 10 5/m l的细胞密度接种 ,接种 2 4 h后换液 ,继代种植密度为 6 .4～ 2 5 .6× 10 4 /ml时最适宜细胞生长 ;在此条件下 ,细胞快速增殖 ,传代后 8h贴壁达 90 %以上 ,2 4 h便进入对数生长期 ,直至第 5 d,细胞约增殖 3倍。本研究建立了在体外分离培养骨髓间充质干细胞的细胞生物学方法和技术参数。     

Simultaneous immunoenzymometric assay for antibodies against human interleukin-2 and human serum albumin in rat serum

A simultaneous immunoenzymometric assay for anti-human interleukin-2 antibody and anti-human serum albumin antibody in rat serum was developed. Two antigen-immobilized polystyrene balls were immersed in a diluted serum sample in an assay tube and then the antibodies on the balls were made to react with a horseradish peroxidase labelled anti-rat IgG antibody in the same tube after washing. The enzyme activity of each ball was measured by fluorometry. Not only were the sensitivity (70 ng/ml each), assay recovery (100-101%), and precision (C.V. = 5-13%) comparable to those of conventional immunoenzymometric assays using one antigen-immobilized ball but the assay was also much more feasible for mass routine assays. Thus, conventional immunoassays can be replaced by this convenient simultaneous method.     

A micro double capillary method for rheologic measurements of lower airway secretions

We modified the double capillary method of Philipoff et al. [23] to allow rheologic measurements of lower respiratory secretions. The system consists of a precision bore stainless steel capillary (0.58 mm i.d., 5 mm length), a glass capillary (0.92 mm i.d., 15 mm length) and a sample holding tube (1.5 mm i.d., 15 mm length). This series of capillaries is connected to a long polyethylene catheter which permits advancing the system through the inner channel of a fiberoptic bronchoscope, and the placing of the sample holding tube into the lower airways for the aspiration of secretions under direct vision. The required sample volume is 20 microliter. The capillary is then removed from the fiberoptic bronchoscope and placed on a microscope stage; the movement of the meniscus of the sample under an applied vacuum as well as the elastic recoil of the sample after release of the vacuum are measured at body temperature. From this procedure, apparent viscosity (na) and shear elastic modulus (G) are calculated. The accuracy of the method was verified in vitro by using standard viscosity oils and a polymer solution of known elasticity. In five normal conscious sheep, na ranged between 200-3000 poises (shear rate: 0.06-1.0 s-1) and G between 17-83 dyn X cm-2. The micro double capillary method was found adequate for direct rheologic measurements of lower airway secretions.     

A new method for bone marrow cell harvesting

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new "perfusion method" for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4(+) and CD8(+) T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this "perfusion method" are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the "perfusion method" is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-host-disease in allogeneic BM transplantation.     

Methodological and clinical evaluation of a microplate assay of endotoxin in whole blood sample

We have developed a simple, rapid method for determining endotoxin in whole blood using a microtiter plate. The samples were pretreated with 0.66 mol/l nitric acid containing 0.25% Triton X-100; the supernatants were placed in a microtiter plate well, and incubated with endotoxin-specific chromogenic limulus test reagent (Endospecy; Seikagaku Corporation). The calibration curve was linear from 0 to 50 pg/ml of Escherichia coli 0111: B4 endotoxin with a variation coefficient less than 5%. The recovery of endotoxin added to human and experimental animals was about 100% without being affected by dilution. The normal endotoxin level in human whole blood was less than 10 pg/ml in reference to E. coli 0111: B4 endotoxin. The results in 120 patients with various diseases correlated well with those determined by a conventional method using plasma as a sample (r = 0.877; p less than 0.05). Whole blood sample has advantages over plasma in that it allows all endotoxin in a sample to be determined, requires a smaller amount of sample, and has less risk of contamination.     

Application of Pop-off Method to Bone Marrow and Peripheral Blood Specimens for Purposes of Electron Microscopy

In this paper, we describe a pop-off method applicable to the hematological field. Bone marrow or peripheral blood specimens from patients were placed on a clean glass slide and fixed immediately in 2% glutaraldehyde solution for 10 minutes. For the DAB reaction, the slide was immersed in the DAB reagent for 30 minutes, and post fixed with 1 % OsO₄ solution for 1 hour. Specimens on the slide were washed with buffer solution, dehydrated and polymerized directly on the slide. A gelatin capsule filled with Epon mixed monomer was then reversed over the specimen. After polymerization was completed, the specimen was popped off from the slide to the capsule and trimmed carefully to prepare for ultrathin sectioning. This method allows the entire sequence of tissue preparation to be carried out on the slide, from fixation to embedding, and even, especially in scanty specimens, including the DAB reaction. Electron microscopic findings in specimens prepared by this technique show excellent preservation and the absence of specific artifacts.     

Application of Pop-off Method to Bone Marrow and Peripheral Blood Specimens for Purposes of Electron Microscopy

In this paper, we describe a pop-off method applicable to the hematological field. Bone marrow or peripheral blood specimens from patients were placed on a clean glass slide and fixed immediately in 2% glutaraldehyde solution for 10 minutes. For the DAB reaction, the slide was immersed in the DAB reagent for 30 minutes, and post fixed with 1 % OsO₄ solution for 1 hour. Specimens on the slide were washed with buffer solution, dehydrated and polymerized directly on the slide. A gelatin capsule filled with Epon mixed monomer was then reversed over the specimen. After polymerization was completed, the specimen was popped off from the slide to the capsule and trimmed carefully to prepare for ultrathin sectioning. This method allows the entire sequence of tissue preparation to be carried out on the slide, from fixation to embedding, and even, especially in scanty specimens, including the DAB reaction. Electron microscopic findings in specimens prepared by this technique show excellent preservation and the absence of specific artifacts.     

Immuno-ultracentrifugation: a combination of density gradient centrifugation and immunodiffusion.

By incorporating acrylamide in a sucrose gradient and photopolymerizing the tubes at the end of the run, direct immunochemical and other analysis can be performed on the separated material in situ without recovering fractions from the centrifuge tube. Immunodiffusion performed on the sliced gel can be employed to locate antibody activity in the gel slice or determine the distribution of antigens in a sucrose gradient     

大鼠骨髓源性内皮祖细胞的分离培养与鉴定

背景：内皮祖细胞因其分离与培养的方法各不相同,在实验中难以重复。 目的：探讨大量获取骨髓源性内皮祖细胞分离与培养的方法。 方法：通过密度梯度离心法从4周龄SD大鼠骨髓中分离单个核细胞,使用EGM-2 MV培养基进行诱导培养,采用形态学特征观察、摄取Dil-Ac-LDL与结合FITC-UEA-1实验、免疫荧光化学鉴定其表面抗原CD133与VEGFR2等方法对其进行鉴定,并通过管腔形成实验观察形成管腔的能力。 结果与结论：①形态学观察：分离的骨髓单个核细胞经诱导培养后,在生长的早期(8 d左右)、晚期(15 d左右)其细胞形态有一定差异,早期以纺锤形、三角形、圆形细胞多见,晚期以圆形、短梭形细胞多见。②摄取Dil-Ac-LDL与结合FITC-UEA-1实验：显示8,21 d的细胞均为阳性。③免疫荧光化学染色：8 d的细胞表达CD133、VEGFR2。④管腔形成实验：在Matrigel基质上15 h左右能够生成血管样结构。结果表明：利用密度梯度离心法分离大鼠骨髓单个核细胞后以EGM-2 MV进行诱导培养,经过鉴定证明获得的细胞符合内皮祖细胞的特征。这种方法能够简单、快速、可靠、大量地获取内皮祖细胞。     

Purification of Japanese encephalitis virus vaccine by zonal centrifugation.

A large-scale procedure for purification and concentration of Japanese encephalitis virus vaccine by a continuous-flow isopycnic banding technique in a sucrose density gradient solution, using a K-III zonal centrifuge rotor, is presented. The quality of zonal-purified vaccine was compared with commercial and Japanese National Institutes of Health reference vaccines for antigenicity, immunodiffusion, and allergic encephalitogenicity tests to show its high purity.     

A direct bone marrow chromosome technique for acute lymphoblastic leukemia

We describe a direct bone marrow chromosome technique that was developed especially for use in studies of acute lymphoblastic leukemia (ALL). The features responsible for technical improvements include: the use of RPMI 1640 medium, supplemented with 30% fetal calf serum, to support cellular activity during both specimen transport and Colcemid treatment; the processing of only 0.1 ml of sedimented cells or less per centrifuge tube; the exposure of cells to Colcemid for a maximum of 25 min; control of the total time of exposure to hypotonic solution; the use of a steel wire as a stirring rod (fashioned to fit the centrifuge tube) for mixing cells; slide preparation by a specific edging-flaming technique; the natural aging of the slides to achieve optimal drying; and the use of a modified G-banding procedure that employs Wright's stain. This technique has been used in more than 350 cases of ALL and has consistently provided analyzable banded chromosomes, even in hyperdiploid cases with up to 91 chromosomes. It makes the previously recognized morphological difference between metaphases of residual normal cells and those from the leukemic clone less apparent. The edging-flaming technique of slide preparation is the most important component and is especially appropriate for spreading large numbers of chromosomes in ALL.