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Safety in the Laboratory
Published in Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts, Retroviral Testing, 2020
Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts
Lab coats and gloves must be used in the contaminated area when handling any equipment, pipettes, specimens, refrigerators, and freezers where samples are stored. Protective equipment must also be used when working under a hood (class II biosafety cabinet). Gloves and laboratory coats should be removed, and hands washed before handling any clean equipment such as the computer, calculator, or telephone. A policy that limits access to laboratory testing areas can be instituted by indicating that “authorized staff only” be admitted; this will significantly limit unauthorized pedestrian traffic, thus avoiding unnecessary contact of individuals with contaminated equipment.
“Kidney in a Dish” Organoids for PKD
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Nelly M. Cruz, Benjamin S. Freedman
Step-by-Step ProtocolRecline the plate at a 45° angle in the cell culture hood or biosafety cabinet. The plate can be resting on top of a 50 mL serological pipet, for example, to maintain the desired angle for 5 min. After 5 min, all the organoids will collect at the bottom edge of the well due to gravity.Use a P1000 to manually remove media from the plate and discard, avoiding the organoids. Leave ∼500 μL media in the plate.Slowly add 2 mL of fresh RB media, drop by drop to not disturb the organoids. Rock the plate back and forth gently to disperse the organoids and put back in the 37°C incubator.
Viral disease and assisted reproduction technology
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Carole Gilling-Smith, Pietro Vernazza
Despite previously published concerns regarding the handling and freezing of gametes and embryos from patients who carry blood-borne viruses (111–113), there have been no reported cases of cross-contamination in the ART setting. Published guidelines on the matter remain limited (111), and for this reason, many ART centers will not treat patients with a known viral infection. The European Society of Human Reproduction and the Embryology Committee of the Special Interest Group on Embryology published guidelines in 2008 on good practice in laboratories. They recommended that gametes and embryos from patients infected with HIV, HBV, and HCV be handled in a dedicated laboratory space at allocated times and processing of these samples within a biosafety cabinet to minimize the risk of cross-contamination of patient specimens. The Practice Committee of the American Society for Reproductive Medicine issued guidance in 2013 recommending that samples from viral-positive patients be handled separately in time or space and that separate cryostorage facilities be used when freezing samples. In the U.K., the HFEA mandates separate cryostorage for gametes and embryos with different viral infections or infection combinations. What is clear from reviewing the published literature is that there are no universally agreed guidelines. The emergence of new viruses such as the Zika virus exemplifies the importance of treating all samples as potentially infectious and using universal precautions at all times, as is used in operating theaters and emergency rooms. There is no difference between handling a sample from a known virally infected patient and a sample from a patient who screened negative for HIV, HBV, and HCV three months before, but might have seroconverted due to a casual relationship in the interim.
Differentiation of rat bone marrow mesenchymal stem cells into neurons induced by bone morphogenetic protein 7 in vitro
Published in Neurological Research, 2023
Heng Zhang, Lei Gao, Wen Zhang, Kuanxin Li
Two SD rats were randomly selected and then weighed and recorded. The rats were sacrificed by cervical dislocation. The femur and tibia were separated by tissue scissors, soaked in 75% alcohol for 5 min and placed in PBS until use. The following operations were performed in a biosafety cabinet. The femoral and tibia were removed with tissue scissors to expose the medullary cavity, and the bone marrow cavity was drained with a 2.5 ml syringe until the bones were transparent. The cell suspension was collected and beat repeatedly. The cell counting plate was carried out to adjust the cell density to 1 × 107 cells/ml to culture in a culture flask. The complete medium was configured to be 89% DMEM (ThermoFisher, MA, USA) + 10% fetal bovine serum (FBS, Sangon Biotech, Inc., Shanghai, China) + 1% 2 mM L-glutamine and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) (Sangon Biotech, Inc., Shanghai, China). The cells were cultured for 48 h in a humidified incubator at 37°C in 5% CO2, and then the whole amount of the medium was changed every 3 days. When the cells were fused to about 80%, they were digested with 2.5 mg/ml trypsin, subcultured at a ratio of 1∶2, and cultured to the third passage.
Drug resistance profiles and related gene mutations in slow-growing non-tuberculous mycobacteria isolated in regional tuberculosis reference laboratories of Iran: a three year cross-sectional study
Published in Pathogens and Global Health, 2023
Sousan Akrami, Azar Dokht khosravi, Mohammad Hashemzadeh
All experiments were performed in Tuberculosis Reference Laboratory in a special and self-contained room physically separated from other parts under the supervision of trained staff. A calibrated autoclave was available for decontaminating laboratory waste. Directional air flow was maintained by extracting room air. All procedures were undertook under a Class II biosafety cabinet. Gloves and masks were used during the experiments. In each run of culture medium preparation or antibiotic susceptibility experiment, the QC strains were used. Subcultures of working QC strains were performed weekly or whenever susceptibility testing was conducted. Storage of these stock cultures was carried out at –80°C in tryptic soy broth with 15% glycerol. When a new batch of test medium was prepared, QC strains were used. Also, there was a free antimicrobial medium with QC strains in each microdilution broth tray to verify viability of the test organisms.
Ready to go 3D? A semi-automated protocol for microwell spheroid arrays to increase scalability and throughput of 3D cell culture testing
Published in Toxicology Mechanisms and Methods, 2020
Amrita Basu, Aneta Dydowiczová, James E. Trosko, Luděk Bláha, Pavel Babica
Hydrogels were prepared from molecular biology grade agarose (Sigma-Aldrich, Cat. No. A9539, CAS No.: 9012-36-6), with no detectable DNase or RNase activity, gel point 36 °C (at 1.5% gel) and gel strength ≥1200 g/cm2 (at 1% gel). A 2% (w/v) agarose solution in a 0.9% sodium chloride was prepared and autoclaved. All further work was conducted aseptically in a biosafety cabinet sterilized with UV-light and 70% ethanol. Sterile molten agarose (kept at 60-70 °C on thermoblock in the biosafety cabinet) was aseptically pipetted into autoclave-sterilized 3D Petri Dish® micromolds (Sigma-Aldrich). Two types of micromolds were used to prepare hydrogels fitting 24-well plates: a) hydrogels with an array of 5 × 7 microwells (recesses), each with 800 µm diameter and 800 µm depth, Cat. No. Z764051, producing 5 × 7 = 35 spheroids per well and 5 × 7 × 24 = 840 spheroids per 24-well plate; or b) hydrogels with an array of 8 × 12 microwells, each with 400 µm diameter and 800 µm depth, Cat. No. Z764043, producing 8 × 12 = 96 spheroids per well and 8 × 12 × 24 = 2304 spheroids per 24-well plate. Unless indicated otherwise, data presented in this study were obtained using hydrogels with 5 × 7 microwell array.