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Bronchoalveolar Lavage in Inhalation Lung Toxicity
Published in Jacob Loke, Pathophysiology and Treatment of Inhalation Injuries, 2020
K. Randall Young, Herbert Y. Reynolds
The optimal method for enumerating different cell populations in BAL has been the topic of some debate (Crystal et al., 1986). Differential counting of Wright-Giemsa-stained cytocentrifuge preparations has been the standard in most laboratories. However, Saltini and co-workers (1984) noted that the proportion of small cells in the original BAL cell suspension was greater than the percentage of lymphocytes identified on cytocentrifuge preparations, and they developed a method for quantitating cell populations collected on Millipore filters and stained with hematoxylin-eosin. Using both filter and cytocentrifuge methods, they examined BAL fluid from 10 normal volunteers and 39 patients with interstitial lung disease. They found that the percentage of lymphocytes identified on cytocentrifuge preparations was lower than that on filter preparations in 88% of the 49 individuals studied. On average, the proportion of lymphocytes determined from cytocentrifuge preparations was 73% of that quantitated on filter preparations. The filter technique was validated on cell mixtures of alveolar macrophages and highly purified lymphocytes and in that setting yielded values that closely approximated the predicted value. The cytocentrifuge method, in contrast, underestimated the percentage of lymphocytes by an average of 36%, with apparent losses of lymphocytes of up to 50% in some cases. These authors also noted that repeated “washing” and centrifugation of cells led to an average loss of 22% of cells present in the original lavage suspension.
Peripheral Blood and Bone Marrow
Published in Harold R. Schumacher, William A. Rock, Sanford A. Stass, Handbook of Hematologic Pathology, 2019
Fermina Maria Mazzella, Gerardo Perrotta
Under special circumstances when body fluids other than blood are being examined, a cytocentrifuge is necessary to concentrate the small numbers of cells to be identified. A monolayer of cells is concentrated on a small circular area of a glass slide.
Chorioretinal biopsy
Published in A Peyman MD Gholam, A Meffert MD Stephen, D Conway MD FACS Mandi, Chiasson Trisha, Vitreoretinal Surgical Techniques, 2019
Slides were prepared using the cytocentrifuge after resus-pending cells in 4 ml of medium. Two slides from each method were stained by Gram, Giemsa, or DiffQuick stains for cytology. The remaining slides were fixed by submersing in acetone for 7 minutes; immunohistochemical staining was performed using the avidin–biotin immunoperoxidase technique. Monoclonal antibodies were used as indicated in Table 44.1.
Modulatory effect of myricitrin against chromosome instability and cytostasis induced by bleomycin and oxaliplatin in CHO-K1 cells
Published in Drug and Chemical Toxicology, 2023
Ana Paula de Souza, Raíne Fogliati Schardosim, Juliana Escouto Al Kateeb, Mauricio Lehmann, Ivana Grivicich, Rafael Rodrigues Dihl
To investigate the mutagenic effect of MYR, two independent experiments were performed in duplicate on different days to ensure reproducibility. After incubation, cells were treated at different concentrations (4.8 µg/mL, 9.7 µg/mL, 19.5 µM, 39 µg/mL and 78 µg/mL) of MYR as well as negative control (DMSO 1%) and positive control (BLM 3 µg/mL) for short (4 h) and extended (24 h) treatment. After treatment, CHO-K1 cells were washed twice in Dulbecco’s phosphate-buffered saline (DPBS, Sigma-Aldrich, St. Louis, MO) and Cyt-B (2.5 µg/mL) was added during two cell cycles. The cells were collected and harvested by cytocentrifugation (Cientec, Belo Horizonte, MG, Brazil); 160 µL of cell suspension were transferred to cytocentrifuge cups and were centrifuged for 5 min at 700 rpm to produce 1 spot per slide. Slides with the cells were stained with Instant Prov (Newprov, Pinhais, PR, Brazil). After staining, slides were air dried and examined under 400x magnifications using a light microscope. To evaluate chromosomal instability, micronuclei (MNi), nuclear buds (NBUDs), and nucleoplasmic bridges (NPBs) were scored in 1000 binucleated cells (BNC) per experimental point, according to Fenech (2007).
How relevant are in vitro culture models for study of tick-pathogen interactions?
Published in Pathogens and Global Health, 2021
Cristiano Salata, Sara Moutailler, Houssam Attoui, Erich Zweygarth, Lygia Decker, Lesley Bell-Sakyi
A. marginale cultures are initiated from infected bovine blood, collected during ascending bacteremia. Culture flasks containing growing layers of IDE8 or ISE6 cells are inoculated with infected blood stabilates, sealed and incubated at 32–34°C with weekly medium changes [109,110]. Initially, compact colonies are observed inside well-defined parasitophorous vacuoles; two or three weeks later, large colonies are formed, and their contents are released into the culture medium after disruption of the vacuole and cell membranes. A. marginale-infected cells can be propagated continuously by serial passage onto naïve tick cells, and can reach infection rates up to 80%, retaining their infectivity and antigenic properties after successive passages [109,113]. Cultured cells can be monitored by direct examination under an inverted microscope and/or by microscopic examination of Giemsa-stained cytocentrifuge smears (Figure 4A).
The sp3/sp2 carbon ratio as a modulator of in vivo and in vitro toxicity of the chemically purified detonation-synthesized nanodiamond via the reactive oxygen species generation
Published in Nanotoxicology, 2020
Dong-Keun Lee, Sangwook Ha, Soyeon Jeon, Jiyoung Jeong, Dong-Jae Kim, Seung Whan Lee, Wan-Seob Cho
At 24 h and 4 weeks after a single intratracheal instillation, the rats were anesthetized with isoflurane (Piramal Critical Care) and sacrificed by taking blood from the abdominal vein. Then, the trachea was intubated using a blunt 14-gauge stainless-steel needle via a small incision in the upper part of the trachea and tightly tied using an elastic suture. Then, the lung was lavaged 4 times with cold sterile PBS. The first lavage was centrifuged at ×2000 g for 5 min to collect cell-free supernatant for the measurement of lactate dehydrogenase (LDH), total protein, and pro-inflammatory cytokines. The cell pellets from the four lavages were pooled and resuspended with 1 mL of sterile PBS. The number of total nucleated cells in the BALF was counted using a NucleoCounter (Chemometec, Allerod, Denmark). Then, cytospin slides were prepared by attaching 4 × 104 nucleated cells using a cytocentrifuge (Hanil, Seoul, Korea). The slides were dried, fixed for 5 min with absolute methanol, and stained with a Diff-Quik staining kit (Thermo Fisher Scientific). Finally, the differential cell counting was performed by counting at least 300 cells per slide using a light microscope (Nikkon, Tokyo, Japan).