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Nucleated Cell Separation Using the Fenwal CS3000™
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Herbert M. Cullis, Ellen Areman, Charles S. Carter
In this procedure, the cells will be left in the centrifuge chamber, about half the plasma will be removed from them, and they will be resuspended in normal saline, and the centrifuge started. With the centrifuge rotating, Ficoll will be pumped into the chamber where it will accumulate on the centrifugal bottom of the chamber. The Ficoll will be pumped in slowly enough to permit the heavier cells to sediment through the gradient material. The lighter cells will be floated to the top of the chamber and will be displaced out of the chamber and will be collected in a bag outside the centrifuge.
Semen Analysis and Sperm Washing Techniques
Published in Claude Gagnon, Controls of Sperm Motility, 2020
These techniques are all based on the use of density-gradient centrifugation to fractionate subpopulations of spermatozoa. Gradient materials like sucrose or caesium chloride are far too osmotically active to be used with live cells such as spermatozoa. Sodium metrizoate*** was initially evaluated as a material for separating human spermatozoa,44 but has not seen wide use. Ficoll† has been used as a gradient material for preparing spermatozoa,45 but by far the most widely used has been Percoll.† Finally, Nycodenz‡ is the latest substance to be used as a density gradient material for preparing human spermatozoa.
Exercise and Neutrophil Activity: A Possible Neuroendocrine Connection
Published in Alan J. Husband, Psychoimmunology CNS-Immune Interactions, 2019
Maurice J. Weidemann, John A. Smith, A. Bon Gray, David B. Pyne, Marysia Kolbuch-Braddon, Richard D. Telford
Ferricytochrome-C (purified from horse heart), luminol (5-amino-2,3-dihydro-1,4-phthazine dione), paraformaldehyde, phorbol myristate acetate (PMA), superoxide dismutase and zymosan-A were all purchased from Sigma (St. Louis, MO). Ficoll-paque was obtained from Pharmacia (Uppsala, Sweden), Mono-poly resolving medium from ICN Biomedicals (Sydney, Aust) and 2,7-dichlorofluorescin diacetate from Serva (Heidelberg, Germany). The fluorochrome-conjugated monoclonal antibodies against complement receptor CR 3 (CD 11b), Fc (Type III) receptor (CD 16), control fluorochrome-conjugated IgG antibodies and FACSLYSE® were purchased from Becton Dickinson (San Jose, CA, USA).
Cancer-specific type-I interferon receptor signaling promotes cancer stemness and effector CD8+ T-cell exhaustion
Published in OncoImmunology, 2021
Wang Gong, Christopher R. Donnelly, Blake R. Heath, Emily Bellile, Lorenza A. Donnelly, Hülya F. Taner, Luke Broses, J. Chad Brenner, Steven B. Chinn, Ru-Rong Ji, Haitao Wen, Jacques E. Nör, Jie Wang, Gregory T. Wolf, Yuying Xie, Yu Leo Lei
Immune cells from tumors and spleens were purified as we have previously described.10 Briefly, tumors were excised from mice and minced into pieces, followed by dissociation by passing through a 70-μm cell strainer to obtain a single-cell suspension. Spleens were processed by mechanical dissociation, followed by lysis of red blood cells (A10492-01, Gibco). Ficoll-Paque PLUS (17–1440-03, GE Healthcare Life Sciences) was added to the bottom of each tube containing single cell suspensions in RPMI1640, followed by density-gradient centrifugation to purify immune cells. Purified immune cells were stained for multi-fluorophore flow cytometric analysis with the following surface antibodies: anti-CD45 (clone 30-F11, BioLegend), anti-CD3 (clone 17A2, BD Biosciences), anti-TCRβ (clone 56–6.7, BioLegend), anti-CD4 (clone RM4-5, BioLegend), anti-CD8 (clone 53–6.7, BioLegend), anti-CD44 (clone IM7, BioLegend), anti-CD366 [Tim3] (clone RMT3-23, Biolegend), and anti-PD-1 (clone 29F1A12, Biolegend). The Cyto-Fast™ Fix/Perm Buffer Set (Cat # 426803, Biolegend) was used for fixation and permeabilization and anti-mouse IFNγ (clone XMG1.2, BioLegend) antibody was used for intracellular staining. Cell viability was measured using Fixable Viability Dye (FVD) eFluor 780 (65–0865-14, Thermo Fisher Scientific) or Zombie Aqua (423101, BioLegend) diluted 1:1000 in PBS at 4°C for 30 minutes. Acquisition and compensation were conducted on a Beckman Coulter CytoFLEX flow cytometer. FlowJo version 10 software was used for data analysis.
Multifaceted applications of pre-mature chromosome condensation in radiation biodosimetry
Published in International Journal of Radiation Biology, 2020
Usha Yadav, Nagesh Nagabhushana Bhat, Kapil Bansidhar Shirsath, Utkarsha Sagar Mungse, Balvinder Kaur Sapra
Blood samples were collected from healthy 25–40 years male and female donors in compliance with the Institutional Ethical Committee. Peripheral blood lymphocytes were separated using Ficoll based density gradient centrifugation. For each fusion, ∼2 million lymphocytes were used per sample. Isolated lymphocytes were irradiated with 60Co gamma rays at a dose rate of ∼1 Gy/min (Gamma chamber-220 facility, at Bhabha Atomic Research Centre, Mumbai, India). The irradiated lymphocytes were incubated in a humidified CO2 incubator at 37 °C at five different time points (0–24 h) to analyze the kinetics of chromosome fragments formation and persistence after 1, 4 and 8 Gy doses. For radiation dose response curve studies, constant 24 h incubation time was given at nine dose points in the range of 0–15Gy.
A novel human monoclonal antibody specific to the A33 glycoprotein recognizes colorectal cancer and inhibits metastasis
Published in mAbs, 2020
Patrizia Murer, Louis Plüss, Dario Neri
CT26A33.C3 and C51A33.A5 were stained with CFSE dye (BioLegend) as described by the manufacturer’s protocol and seeded in a 48-well plate (2⋅104/well). After informed consent was given, peripheral blood samples were obtained from healthy donors from the Blutspendedienst SRK, Zurich. PBMCs were isolated by density gradient centrifugation on Ficoll Paque Plus (GE Healthcare) following the manufacturer’s protocol. The peripheral blood was diluted 1:3 in 2 mM EDTA/PBS. Thirty milliliters of diluted peripheral blood was layered on 12.9 ml Ficoll and centrifuged at 400 g for 40 min at RT. PBMCs were collected, washed twice with PBS/EDTA, and incubated with the tumor target cells (106/well, 1:50 target:effector ratio) in CT26A33.C3 and C51A33.A5 culture media. The antibody IgG1(A2) was added at different concentrations to the wells (total volume of 200µl/well) and the plate was incubated at 37°C, 5% CO2 for 24 h. On the next day, the samples with IgG1(A2) + PBMCs + target cells were transferred to a 96-well plate, washed twice with PBS and stained Fixable Viability Dye (Invitrogen) for 30 min. The samples were washed twice with 2 mM EDTA 0.5% BSA in PBS, before being analyzed by FACS (CytoFLEX, Beckman Coulter). The percentage of living target cells was derived from the mean fluorescence intensity of the CFSE-positive cell population normalized based on the negative control samples containing PBMCs and target cells only.