Following local anesthesia with nebulized lidocaine and i.v. conscious sedation, subjects underwent flexible bronchoscopy. Briefly, a flexible fiberoptic bronchoscope was inserted transorally and advanced through the vocal cords. Bronchoalveolar lavage (BAL) fluid was obtained from tertiary airways in the right upper lobes (RUL) and right lower lobes (RLL). BAL was performed sequentially in the RUL and RLL with 20 ml of sterile saline followed by 10 ml of air, and this was repeated for a total of five times per airway. Lung macrophages were isolated, as previously described (13 (link), 14 (link)). BAL fluid was filtered through two-layer gauze, centrifuged, and washed twice in 0.9% NaCl. Cells were counted with a T10 automated cell counter (Bio-Rad, Hercules, CA). Cytospins were performed using a Shandon Cytospin 3 centrifuge (Thermo Fisher Scientific, Waltham, MA). Briefly, 75,000 cells resuspended in 200 μl of 0.9% NaCl were loaded into a cytology funnel (Fisher Scientific, Pittsburgh, PA) and centrifuged for 10 min. Cells were allowed to air dry and processed for viewing via Hema 3 Stat pack (Fisher). Imaging was done on an Olympus (Waltham, MA) BX41 microscope with DP2-BSW software (version 2007).
Shandon cytospin 3 centrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Shandon Cytospin 3 is a centrifuge designed for the preparation of cell samples for microscopic examination. It operates by concentrating cells onto a glass slide, creating a thin, even layer for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bx41 microscope, by Olympus (2 mentions)
Dp2 bsw software, by Olympus (2 mentions)
Cytology funnel, by Thermo Fisher Scientific (2 mentions)
Hema 3 stat pack, by Thermo Fisher Scientific (2 mentions)
T10 automated cell counter, by Bio-Rad (2 mentions)
Thinprep kit, by Hologic (2 mentions)
Clone 4h84, by BD (2 mentions)
Clone g233, by Exbio (2 mentions)
Preservcyt, by Hologic (2 mentions)
Silane coated glass slides, by Muto Pure Chemicals (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Cd16 32, by BD (1 mentions)
Cd206, by BioLegend (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A11003, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Ab31412, by Abcam (1 mentions)
Vectorshield mounting medium, by Vector Laboratories (1 mentions)
Dynamag spin magnet, by Thermo Fisher Scientific (1 mentions)
Giemsa stain, by Merck Group (1 mentions)
13 protocols using shandon cytospin 3 centrifuge
1
Isolation and Analysis of Lung Macrophages from BAL Fluid
2
Immunofluorescence Labeling of Organelles
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Cells and mitosomes after isolation were fixed with 4% PFA for 15 min and adhered to silane-coated glass slides (Muto Pure Chemicals Co., Tokyo, Japan) using a Shandon Cytospin 3 Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 800 rpm for 10 min. After washing with PBS for 15 min, specimens were immersed in blocking solution (1% BSA, 0.2% saponin in PBS) for 30 min at room, and then reacted with anti-HA-tag-Alexa Fluor 488 (M180-A48, MBL Co., Nagoya, Japan; 1/500 dilution in blocking solution) and/or anti-Myc-tag-Alexa Fluor 594 (M047-A59, MBL Co.; 1/200 dilution in blocking solution) at 4 °C overnight. After washing with 0.1% BSA in PBS for 15 min, glass slides were embedded with 10% glycerol in PBS containing 1.25 mg/ml DABCO and stored at 4 °C in the dark.
3
Macrophage Recruitment and Phagocytosis Assay
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To recruit macrophages into the peritoneum, mice were injected i.p. with 3 ml of 3% thioglycolate. Although thioglycolate treatment associated with a ∼10-fold increase in the number of circulating macrophages, it did not influence their surface expression of leptin receptor (assessed by flow cytometry as MFI, not shown). Contralateral i.p. injection of recombinant mouse leptin (6 consecutive doses of 2 µg/g at 12-h intervals) or vehicle was given starting at the time of thioglycolate treatment. At day 3 post-injection, mice were infused i.p. with 1×107 CFSE-labeled apoptotic Jurkat cells. After 30 min, peritoneal cells were recovered. To remove erythrocytes and non-phagocytosed bodies, cells were incubated on polystyrene dishes for 1 h, and then washed. For determination of macrophage uptake of apoptotic cells (CFSE+) by flow cytometry, collected cells were stained with PE-conjugated anti-mouse CD11b Ab. For confocal microscopy, cells were resuspended in 1% cold BSA/PBS, centrifuged in a Shandon Cytospin 3 centrifuge (Thermo Fisher Scientific, Waltham, MA), and dried sediments were fixed with −20°C acetone, blocked with 3% BSA/PBS, and stained with anti-mouse CD11b Ab before visualization with a LSM 310 laser scanning confocal microscope (Carl Zeiss Inc., Toronto, Canada).
4
Peritoneal Macrophage Profiling in Schistosoma Infection
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5000 live Schistosoma mansoni eggs (obtained from the same source as the cercariae described above) were injected i.p. to prime some mice on day 0 while others were left untreated. On day 14, some of the primed mice were challenged i.p. with 5000 live eggs, and some of the primed mice were left unchallenged. On day 18, peritoneal cells were harvested from each group of mice (naïve, primed/rested, primed/rechallenged). Equal numbers of unsorted peritoneal cells were stained with anti-mouse antibodies against F4/80 (Biolegend), CD11b (eBioscience), CD16/32 (BD), and CD206 (mouse mannose receptor, C type 1) (Biolegend) or with a rat IgG2a isotype control. CD206 fluorescence on F4/80hiCD11bhi macrophages was measured using a BD FACSCanto II flow cytometer and FlowJo v.7.6 software (Tree Star; Ashland, OR). F4/80hiCD11bhi peritoneal cells were also sorted as described above. Some sorted cells were spun for 5 mins with a Shandon Cytospin 3 centrifuge (Thermo Scientific; Waltham, MA) onto a slide before being fixed with methanol and stained with Diff-Quik (Boehringer). Aliquots of 5×105 sorted cells were resuspended in lysis buffer and arginase activity was measured as previously described [39] .
5
Isolation and Analysis of Lung Macrophages from BAL Fluid
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Following local anesthesia with nebulized lidocaine and i.v. conscious sedation, subjects underwent flexible bronchoscopy. Briefly, a flexible fiberoptic bronchoscope was inserted transorally and advanced through the vocal cords. Bronchoalveolar lavage (BAL) fluid was obtained from tertiary airways in the right upper lobes (RUL) and right lower lobes (RLL). BAL was performed sequentially in the RUL and RLL with 20 ml of sterile saline followed by 10 ml of air, and this was repeated for a total of five times per airway. Lung macrophages were isolated, as previously described (13 (link), 14 (link)). BAL fluid was filtered through two-layer gauze, centrifuged, and washed twice in 0.9% NaCl. Cells were counted with a T10 automated cell counter (Bio-Rad, Hercules, CA). Cytospins were performed using a Shandon Cytospin 3 centrifuge (Thermo Fisher Scientific, Waltham, MA). Briefly, 75,000 cells resuspended in 200 μl of 0.9% NaCl were loaded into a cytology funnel (Fisher Scientific, Pittsburgh, PA) and centrifuged for 10 min. Cells were allowed to air dry and processed for viewing via Hema 3 Stat pack (Fisher). Imaging was done on an Olympus (Waltham, MA) BX41 microscope with DP2-BSW software (version 2007).
6
Immunofluorescence Analysis of PEG3, p105/p50, and p100/p52
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TCam-2 cells were harvested after 48 h of transfection for cytospin preparation using Shandon Cytospin 3 centrifuge (Thermo Scientific, Waltham, MA, USA). The cytospun cells were washed with cold PBS twice before fixation with 4% paraformaldehyde (USB Corporation/Affymetrix, Santa Clara, CA, USA) for 10 min at room temperature. Cells were permeabilized with cold solution of 0.5% Triton-X for 10 min followed by overnight incubation at 4 °C with anti-PEG3 (ab139166; Abcam; 1:200), anti-p105/p50 (ab31412; Abcam; 1:50) or anti-p100/p52 (3017S; Cell Signaling Technology; 1:100) primary antibodies. Alexa Fluor 488 and 546-conjugated secondary antibodies (A11008 and A11003, respectively; Life Technologies) were diluted to 1:400 and applied on the cells for an hour at room temperature. Cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) in Vectorshield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured in a confocal microscope Leica TCS SP5 (Lecia Microsystems GmbH, Wetzlar, Germany).
7
Isolation of Trophoblast Cells from Endocervix
8
Cytospin Preparation of Intact Nuclei from FFPE Tissue
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Cytospin preparations of intact nuclei derived from disintegrated archival patient material were performed as described31 (link). Briefly, 3 µm formalin-fixed, paraffin-embedded (FFPE) tissue sections stained with Hematoxylin–Eosin were used to evaluate the morphology and outline a representative tumor area (tumor content 80–100%, tumor area 4 cm2 for resections specimens or biopsy cores of 1–1.5 cm length). Adjacent 50 µm sections of the FFPE archival patient tissue were cut, the representative area was macro-dissected from the sections and disintegrated with 0.1% protease. The resulting single-cell suspensions were used to prepare cytospins (Shandon Cytospin 3 centrifuge, Thermo Scientific, Asheville, NC) yielding intact monolayers with an average of around 15,000 single cells.
9
Endocervical Sampling for Trophoblast Cell Analysis
10
Comprehensive Thyroid Gland Assessment
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The histological type, disease stage, and condition of the regional lymphatic system were assessed before surgery.
Doppler ultrasound of the thyroid gland and all neck areas was performed with an expert-grade Philips N-Visor D-4 ultrasound machine (Netherlands) with a sensor frequency of 10 MHz in real-time. The dimensions, shape, and volume of thyroid tissue were assessed. All pathological formations in the thyroid gland and neck region were detected, and their localization, size, number of nodes, structure and the presence of pathological blood flow were assessed.
Fine-needle aspiration biopsy of the thyroid tumour was performed with a 25G needle under ultrasound control. At least 3 punctures were performed for each formation. Evaluation of all native cytological samples was carried out immediately after the puncture by May- Grünwald-Giemsa staining. Cytospins of sorted cellular populations were prepared by cytocentrifugation at 1000 rpm in a Shandon Cytospin 3 centrifuge for 5 min (Thermo Scientific, USA). The samples were fixed in methanol and stained with May-Grünwald stain for 15 min followed by 30 min in 5% Giemsa stain (Merck, Darmstadt, Germany). Slides were then washed with distilled water, dried, and mounted with Entellan (Merck, Darmstadt, Germany).
Doppler ultrasound of the thyroid gland and all neck areas was performed with an expert-grade Philips N-Visor D-4 ultrasound machine (Netherlands) with a sensor frequency of 10 MHz in real-time. The dimensions, shape, and volume of thyroid tissue were assessed. All pathological formations in the thyroid gland and neck region were detected, and their localization, size, number of nodes, structure and the presence of pathological blood flow were assessed.
Fine-needle aspiration biopsy of the thyroid tumour was performed with a 25G needle under ultrasound control. At least 3 punctures were performed for each formation. Evaluation of all native cytological samples was carried out immediately after the puncture by May- Grünwald-Giemsa staining. Cytospins of sorted cellular populations were prepared by cytocentrifugation at 1000 rpm in a Shandon Cytospin 3 centrifuge for 5 min (Thermo Scientific, USA). The samples were fixed in methanol and stained with May-Grünwald stain for 15 min followed by 30 min in 5% Giemsa stain (Merck, Darmstadt, Germany). Slides were then washed with distilled water, dried, and mounted with Entellan (Merck, Darmstadt, Germany).
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