Bronchoalveolar Lavage in Inhalation Lung Toxicity
Jacob Loke in Pathophysiology and Treatment of Inhalation Injuries, 2020
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.
Cytology of Bladder Cancer
George T. Bryan, Samuel M. Cohen in The Pathology of Bladder Cancer, 2017
The cytocentrifuge prepares cells by centrifuging the specimen onto a glass slide. This technique concentrates cells on a small area of the slide by removing the fluid component of the sample. In the process of fluid removal, cells are frequently also removed. Quantitative studies show that cell recovery for diagnostic analysis may be as low as 10%.26 When preparing samples that are scantily cellular, cell losses will be unacceptable. We refer the reader to descriptions of the cytospin method and details of the procedure by Barrett,27 Bales,10 and Schumann.25
Chorioretinal biopsy
A Peyman MD Gholam, A Meffert MD Stephen, D Conway MD FACS Mandi, Chiasson Trisha in 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).
Inhalation exposure to multi-walled carbon nanotubes alters the pulmonary allergic response of mice to house dust mite allergen
Published in Inhalation Toxicology, 2019
Mark D. Ihrie, Alexia J. Taylor-Just, Nigel J. Walker, Matthew D. Stout, Amit Gupta, Jamie S. Richey, Barry K. Hayden, Gregory L. Baker, Barney R. Sparrow, Katherine S. Duke, James C. Bonner
Bronchoalveolar lavage fluid (BALF) was obtained by lavaging the lungs twice with 0.5 ml sterile DPBS. A Thermo Scientific Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA) was immediately used to spin cells from 100 µl of BALF onto glass slides. These cells were then fixed and stained with the Diff-Quik Stain Set (Dade Behring Inc., Newark, DE, USA). The remaining BALF was stored at −80 °C. After BALF collection, the middle and caudal lobes of the right lung, as well as the heart, spleen, and a section of the liver, were placed in RNAlater (Ambion, Austin, TX), according to the manufacturer’s instructions and stored at −80 °C. The cranial lobe of the right lung was flash frozen in liquid nitrogen and stored at −80 °C. The left lung was infused with 10% neutral buffered formalin, fixed for 24 h, dehydrated in 70% ethanol, and embedded in paraffin. Whole blood was collected from the jugular veins, allowed to coagulate for 15 min in Serum Separator Tubes (BD Microtainer, Franklin Lakes, NJ), then centrifuged to obtain serum. Serum was stored at −80 °C.
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).
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