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Chapter 4: Monodisperse Polymer Particles in Immunoassays and Cell Separation
Published in Alan Rembaum, Zoltán A. Tökés, Micro spheres: Medical and Biological Applications, 2017
Kjell Nustad, Havard Danielsen, Albrecht Reith, Steinar Funderud, Tor Lea, Frode Vartdal, John Ugelstad
Peripheral blood mononuclear cells (PBM) were obtained by Isopaque Ficoll gradient centrifugation.33 In these experiments 106 PBM were rosetted with 2 mg particles corresponding to a particle/cell ratio of approximately 20. Aliquots of PBM (5 × 106 cells/mℓ) were incubated in HBSS-1% FCS with particles (A), (B), and (C), respectively in plastic tubes for 30 min at room temperature. The tubes were carefully rotated for 1 min at 10-min intervals. Before separation, small samples were taken from the particle-cell suspensions and the number of rosette-forming cells was counted (Table 1). Magnetic separation of cells was carried out by letting the particle-cell mixture flow (1 to 2 mℓ/min) through a 3-mm plastic tube attached to a V-shaped iron block lined with cobalt-samarium magnets. Effluent cells were centrifuged and the cell pellet was resuspended in RPMI 1640 and counted on a Coulter DN cell counter. Cells incubated with particles (A), (B), and (C), as well as untreated PBM, were assayed for rosette formation with 2-aminoethyl-isothiouronium bromide-treated sheep erythrocytes (EAET) and for 3H-thymidine incorporation after stimulation with phytohemagglutinin (PHA), pokeweed mitogen (PWM), purified protein derivative (PPD), and irradiated allogeneic PBM as described elsewhere.34 The main results of these experiments are shown in Table 1.
Selection of Material for Dialysis Membrane
Published in Sirshendu De, Anirban Roy, Hemodialysis Membranes, 2017
A blood cell aggregation study was carried out to measure the changes in the surface property of blood cells. Freshly collected blood was centrifuged at 700 rpm and the collected pellet was resuspended with normal saline in 1:9 volume ratios; 100 μL of this solution was mixed with 600 μL of normal saline. For RBC aggregation study, equal sizes of membranes were incubated with prepared suspension for 1 hour at 37°C. A white blood cell (WBC) aggregation study was carried out by isolating WBCs from uncoagulated freshly isolated blood by the Ficoll-Paque mononuclear cell isolation principle using HiSep™ LSM-1077 (Himedia) based on manufacturer’s instructions. They were then mixed with normal saline and incubated with membranes as previously. After incubation, the cell suspension was smeared on a glass slide and observed under the microscope (Axio Observer Z1Carl Zeiss, Germany).
Defining Immunological Properties of Nucleic Acid Nanoparticles Using Human Peripheral Blood Mononuclear Cells
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
Marina A. Dobrovolskaia, Kirill A. Afonin
CRITICAL: Human blood may contain bloodborne pathogens. Therefore, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition, recommends following BSL-2 precautions during blood work. Laboratory staff should wear pants, close-toed shoes, disposable gloves, a laboratory coat, and eye protection. In addition, all procedures with human blood in which splashes or aerosols may be created should be conducted inside a biological safety cabinet. Bloodborne pathogen awareness training may be required and is strongly recommended. Any laboratory personnel working with human-derived blood should refer to the OSHA Bloodborne Pathogen Standard for specific required precautions. Always follow your local and institutional policies for working with human blood and, in case of any concerns or questions, contact your Biosafety Officer (BSO) or Institutional Biosafety Committee (IBC). Obtaining blood from patients with certain types of diseases also requires Institutional Review Board (IRB) approval.Place freshly drawn blood into 15- or 50-mL conical centrifuge tubes separated per donor. Add an equal volume of room temperature PBS and mix well.Use 3 mL of Ficoll-Paque solution per 4 mL of blood/PBS mixture. For example, 15 mL Ficoll-Paque per 20 mL of diluted blood in a 50 mL tube.Slowly layer the Ficoll-Paque solution underneath the blood/PBS mixture by placing the tip of the pipet containing Ficoll-Paque at the bottom of the blood sample tube. Alternatively, the blood/PBS mixture may be slowly layered over the Ficoll-Paque solution.
Pedunculagin isolated from Plinia cauliflora seeds exhibits genotoxic, antigenotoxic and cytotoxic effects in bacteria and human lymphocytes
Published in Journal of Toxicology and Environmental Health, Part A, 2022
Amanda Silva Fernandes, Jefferson Hollanda Véras, Luana Santos Silva, Sara Cristina Puga, Elisa Flávia Luiz Cardoso Bailão, Matheus Gabriel de Oliveira, Clever Gomes Cardoso, Cristiene Costa Carneiro, Suzana Da Costa Santos, Lee Chen-Chen
To obtain the lymphocyte culture, 20 ml whole blood was collected using sterile disposable syringes and placed in heparinized tubes. After collection, 20 ml RPMI medium was added to the blood and subsequently transferred to centrifuge tubes (15 ml) and centrifuged at 120 × g for 4 min at 18°C. After centrifugation, blood plasma was discarded, buffy coat layer removed and deposited into fresh centrifuge tubes containing Ficoll-paque plus (human lymphocyte isolation medium). Further centrifugation was performed at 200 × g for 10 min at 18°C; the remaining plasma was discarded, and the pellet resuspended in 10 ml RPMI medium. The solution was again supplemented with 10% FBS and centrifuged at 200 × g for 5 min at 18°C. After discarding the supernatant, the lymphocytes were resuspended in supplemented RPMI medium, transferred to a 25 cm2 cell culture flask containing 0.5% phytohemagglutinin (mitogenic stimulator) and 0.2% antibiotic solution of penicillin and streptomycin (1:1), and incubated at 37°C in a biological oxygen demand (BOD) incubator for 24 hr.
Design of Ti composite with bioactive surface for dental implant
Published in Materials and Manufacturing Processes, 2020
Kotheril Ashwathy Thomas, Swati Dey, Nashrin Sultana, Koustav Sarkar, Shubhabrata Datta
To study the biocompatibility of the Ti alloy and composites, Peripheral Blood Mononuclear Cells (PBMCs) are isolated from the normal donor blood (under the guidelines of the Institutional Ethics Committee) through density gradient centrifugation using Ficoll-Hypaque solution, as reported earlier.[26,27] Here the collected blood sample was diluted with phosphate buffered saline (PBS) (1:1 dilution) and layered over a Ficoll-Hypaque solution. Then, the solution is spun at 2200 rpm for 30 min before removing the tubes from the centrifuge while not disturbing the layering. The PBMC layer is then removed from the tube and transferred to a new 15 ml conical tube, avoiding aspirating Ficoll-Hypaque solution. The PBMC is washed by adding enough PBS to make up 15 ml, spun at 1500 rpm for 10 min and washed again in PBS. The cells are counted with the hemocytometer and the cell viability is determined with trypan blue by mixing a small volume (10 µl) of the PBMC with trypan blue solution 1:1 in a microtiter plate. The hemocytometer is loaded with the cell mixture and kept for at least 30 s before counting. The counted cells are cultured in 24 well plate at the concentration of 1 × 106 cells/ml of RPMI-1640 with 10% Fetal Bovine Serum (FBS) in presence of Penicillin/Streptomycin antibiotics for 3 days on the specimens. The cells attached or adjoining to the Ti alloy and composite are observed on a regular basis for 3 days using EXI-300 Trinocular Inverted Microscope.
Effects of a resistance-training programme on endoplasmic reticulum unfolded protein response and mitochondrial functions in PBMCs from elderly subjects
Published in European Journal of Sport Science, 2019
Brisamar Estébanez, Osvaldo C. Moreira, Mar Almar, José A. de Paz, Javier Gonzalez-Gallego, María J. Cuevas
Venus blood samples (30 mL) were obtained, in the early morning in the fasted state, from the brachiocephalic vein using the EDTA anticoagulant VacutainerTM systems (BD, Franklin Lakes, NJ, USA), 5–6 days before and after the training period. In the case of the young subjects, the blood samples were collected only once at rest. To avoid circadian effects, all samples were collected between 08:00 and 09:00 am. Participants were required to avoid any intense exercise during the previous 5–6 days. No caffeine or alcohol was allowed during this time. Total blood was centrifuged to isolate plasma and PBMCs using density gradient centrifugation on Ficoll separation solution (Biochrom AG, Berlin, Germany). PBMCs were used as representative cells of the immune system, and also chosen in the interest of practicalities and cost, offering a useful alternative for gene expression analyses as a substitute for tissues that are not easily accessible (Fratta Pasini et al., 2018).