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Cryopreservation of Human Bone Marrow Grafts
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Both the volume of the grafts and the quantity of contaminating cells, such as red cells and mature granulocytes, can be reduced by prior processing. Simple centrifugation can separate a buffy coat fraction which contains the majority of the nucleated cells.10,45,46 Centrifugation through a density gradient, using Ficoll-Hypaque, or Percoll, can further decrease contamination by red cells and mature granulocytes.47–51 These cells do not survive freezing and thawing by the method described, and contribute to undesirable debris in the transfusate.48–52 Reduced “toxicity” has been reported after infusion of Ficoll-separated marrow grafts.12 Blood washing and apheresis devices can be used for this purpose and greatly reduce the time and labor of processing.10,44–50 Recoveries of colony-forming progenitor cells have been reported to be unaffected by cell separation.48
Semen Analysis and Sperm Washing Techniques
Published in Claude Gagnon, Controls of Sperm Motility, 2020
Probably the most practical Percoll-gradient procedure is that developed by Dravland51,59 (Appendix VIII). The yield of this method can be extremely high with normal semen samples, ranging from 15 to 96% of motile spermatozoa applied to the gradient.51 Yield is apparently dependent only upon the motility of the spermatozoa in the original semen sample.60 There is variable morphological selection of spermatozoa in the prepared population, with tail and/or midpiece defects being primarily excluded.
Establishment and Characterization of Cancer Cell Lines for Companion Diagnostics
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Serum-free conditions for the culture of some human colorectal cancer cell lines have been described (Murakami and Masui, 1980), but these conditions are generally not suitable for newly isolated carcinoma cultures, which require serum. Colorectal carcinoma has been cultured from biopsies that have been taken from both primary tumors and metastases (Paraskeva and Williams, 1992; Park and Gazdar, 1996) and have been used as a model for studying the control of epithelial-mesenchymal transition. Some colorectal tumors occur with neuroendocrine properties, and some success has been reported on the use of HITES medium (Lundqvist et al., 1991). Density centrifugation on Percoll has been used to purify colonic carcinoma cells for primary culture in conventional medium (RPMI1640 with 10% FBS) (Csoka et al., 1995).
Low dose gamma irradiation pretreatment modulates the sensitivity of CNS to subsequent mixed gamma and neutron irradiation of the mouse head
Published in International Journal of Radiation Biology, 2021
Alla V. Rodina, Yulia P. Semochkina, Olga V. Vysotskaya, Anastasia N. Romantsova, Aleksandr N. Strepetov, Elizaveta Y. Moskaleva
The procedure was carried out in accordance with the method of Legroux et al. (2015). After the perfusion, the brains were extracted from the skull and the cerebellum and olfactory lobes were removed. The brains from each mouse were placed in cold PBS with sucrose (20 g/L) and glucose (0.9 g/L) and kept on ice at +4 °C. Then the PBS was decanted, the brain was thoroughly chopped with a scalpel in StemPro™ Accutase™ Cell Dissociation Reagent (ThermoFisher Scientific, USA), transferred to a test tube, another 2 mL of accutase was added and incubated in a water bath at +37оС for 15 min with stirring. Afterward, 10% by volume of fetal bovine serum was added to the specimens, which were placed in an ice bath. The tissue homogenate was rubbed through a nylon mesh with a pore size of 100 µm. Centrifuged cells were suspended in 20% Percoll (GE Healthcare, USA) (10 mL per 1 brain). Percoll was layered with Hanks solution and centrifuged. Myelin was present in interphase, while cells were sedimented to the bottom. The myelin layer and supernatant were removed. The cell pellet was washed 2 times with PBS. The cell number was counted in the Goryaev,s chamber.
Morphometric analysis of sperm used for IVP by three different separation methods with spatial light interference microscopy
Published in Systems Biology in Reproductive Medicine, 2020
Marcello Rubessa, Mikhail E. Kandel, Sierra Schreiber, Sasha Meyers, Douglas H. Beck, Gabriel Popescu, Matthew B. Wheeler
Discontinuous Percoll gradients were prepared by combining Sperm-TALP (Tyrode’s Albumin Lactate Pyruvate) and ISO-Percoll. Sperm-TALP-basic-medium (Sattar et al. 2011), was supplemented with pyruvic acid (Sigma-Aldrich, St. Louis, MO) and gentamycin sulfate (Sigma-Aldrich, St. Louis, MO). ISO-Percoll was made by combining sodium bicarbonate with Earle’s Balanced Salt Solution (EBSS) (Thermo Fisher Scientific, Waltham, MA) and vortexing in a tube, then combining with Percoll® (Sigma-Aldrich, St. Louis, MO). The pH was adjusted to 7.4. ISO-Percoll and Sperm-TALP were combined to create 45% and 90% mixture gradients (45% on the top of 90%). The osmolarity of the ISO-Percoll was measured at 297–303 mOSM by freezing point depression. Semen was slowly pipetted down the side of the tube to create a third layer on top of the 45% gradient and then centrifuged at 460 x g for 25 minutes. The supernatant was discarded, and the pellet was washed in one milliliter of Sperm-TALP and centrifuged at 250 x g for 10 minutes. The supernatant was again discarded, and the pellet was suspended in one milliliter of Sperm-TALP and centrifuged at 170 x g for 10 minutes. The supernatant was removed and 50 μl of Sperm-TALP was added to the pellet.
Allogenic chondrocyte/osteoblast-loaded β-tricalcium phosphate bioceramic scaffolds for articular cartilage defect treatment
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Shuai Wu, Zhiguo Kai, Dong Wang, Lina Tao, Peng Zhang, Dawei Wang, Dongxing Liu, Shui Sun, Jian Zhong
Percoll diluted solution was prepared by mixing 9 volume of Percoll solution (Pharmaeia, Uppsala, Sweden) and one volume of 10X PBS. Complete cell culture medium consists of 90% low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM, HyClone Inc, USA), 10% fetal bovine serum (FBS; Gibco, Carlsbad, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. Osteoblast induction solution was prepared by adding dexamethasone (100 nM), β-glycerol phosphate sodium (10 mM), and vitamin C (0.05 mM) into Hepes-buffered Dulbecco’s modified Eagle’s medium (H-DMEM, HyClone Inc, USA) with 10% FBS. Chondrocyte induction solution was prepared by adding bone morphogenetic protein-2 (BMP-2, 200 ng/mL), insulin-like growth factor 1 (IGF-1, 10 ng/mL), transferrin (6.25 μg/mL), dexamethasone (0.1 μM), and vitamin C (50 μg/mL) into H-DMEM with 10% FBS.