To induce acute pulmonary inflammation we adapted the described model of LPS-induced ALI [27 (link)]. Briefly, LPS from E. coli 0111:B4 (10 μg LPS/50 μL PBS; Sigma-Aldrich, Taufkirchen, Germany) was instilled intratracheally during anaesthetic inhalation of isoflurane (Baxter, Unterschleißheim, Germany) to trigger neutrophil infiltration into the lung. PBS served as negative control. 100 U PC/kg was administered intravenously 0.5 h after LPS installation in WT mice or RAGE- or Icam-1-deficient mice to dissect the role of RAGE and ICAM-1. Six hours after LPS application mice were anesthetized by i.p. injection as already mentioned, the trachea was cannulated and a bronchoalveolar lavage (BAL) of the right lung was performed using a rinse solution containing PBS and protease inhibitor solution (Protease Inhibitor Cocktail, Sigma-Aldrich, Taufkirchen) to harvest infiltrated cells. For leukocyte differentiation, cells were coated on microscopic slides using a Shandon Cytospin Centrifuge (Thermo Fisher Scientific, Waltham, USA), stained with Giemsa/May Grünwald solution, and analyzed on a Leica DMRB upright microscope and a ×100/0.75 NA oil immersion objective (both from Leica, Wetzlar, Germany).
Shandon cytospin centrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Shandon Cytospin centrifuge is a laboratory instrument designed for the preparation of cell samples for microscopic examination. It utilizes centrifugal force to deposit cells onto a microscope slide, enabling the assessment of cellular morphology and other diagnostic tests.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Dmrb upright microscope, by Leica (1 mentions)
Isoflurane, by Baxter (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Optiphot, by Nikon (1 mentions)
Tc10 automated cell counter, by Bio-Rad (1 mentions)
Wright giemsa, by Thermo Fisher Scientific (1 mentions)
Neubauer haemocytometer, by Merck Group (1 mentions)
7 protocols using shandon cytospin centrifuge
1
Acute Pulmonary Inflammation Induction by LPS
2
Apoptotic Cell Identification via PI Staining
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Apoptotic
cells were identified
in propidium iodide (PI) stained cells by morphology.19 (link) At the end of the experiment, cells were spined to slides
using a Shandon cytospin centrifuge (Thermo Scientific, Waltham, MA,
U.S.A.) and fixed in a solution containing 50% acetone and 50% methanol.
The cells were then stained with PI at a concentration of 1 μg/mL.
A microscope (Nikon Optiphot, Tokyo, Japan) was used to identified
apoptotic cells according to the morphological changes characteristic
of apoptosis as described earlier.19 (link)
cells were identified
in propidium iodide (PI) stained cells by morphology.19 (link) At the end of the experiment, cells were spined to slides
using a Shandon cytospin centrifuge (Thermo Scientific, Waltham, MA,
U.S.A.) and fixed in a solution containing 50% acetone and 50% methanol.
The cells were then stained with PI at a concentration of 1 μg/mL.
A microscope (Nikon Optiphot, Tokyo, Japan) was used to identified
apoptotic cells according to the morphological changes characteristic
of apoptosis as described earlier.19 (link)
3
Quantification of BrdU Incorporation with Dactolisib Treatment
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Cells exposed to 0, 10, 50, 250 and 1000 nM dactolisib for 72 h, were treated with 10 μM BrdU (Sigma-Aldrich, St. Louis, MO, USA) in medium for 45 min at 37 °C. They were detached using a cell scraper, washed once with 1xPBS and resuspended to a concentration of 1 × 105 cells/ml. Cell suspensions were kept on ice and processed within minutes. One hundred microliter cell suspension from each sample was loaded into individual sample chambers and centrifuged in a Shandon CytoSpin centrifuge (Thermo Fisher Scientific, Wilmington, DE, US) at 800 rpm for 3 min. Immobilized cells were fixed (described in the ICC-section below), and subsequently subjected to immunocytochemistry, imaging and quantification. For each slide, three randomly picked areas (832 μm × 665.6 μm, 554 mm2) were selected for quantification. The FITC stained cells and the total number of cells was manually counted, and the proportion of FITC positive cells was calculated.
4
Kinetochore Pair Distance Measurement
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Indicated cells were seeded in a 6‐well plate, RNAi knockdown was performed, and cells were synchronized using thymidine followed by nocodazole treatment. 48 h after RNAi transfection, mitotic cells were collected by shake‐off. After hypotonic treatment with KCl, cells were spun onto microscopy slides with a Shandon Cytospin centrifuge (Thermo Fischer), fixed with 4% paraformaldehyde, and then immunocytochemistry was performed. Representative images were taken with a 100× objective on a DeltaVision fluorescent microscope under the same condition. The distance between the two peak intensities of CREST or YFP‐CB was measured for 5 kinetochore pairs using ImageJ and averaged for a single cell. At least 45 cells from minimum of 3 independent experiments were analyzed.
5
BrdU Incorporation Assay for Proliferation
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Cells exposed to buparlisib for 72 h were treated with 10 µM BrdU (Sigma-Aldrich) for 45 min at 37 °C. They were detached using a cell scraper, washed with 1xPBS and resuspended to 1 × 105 cells/mL. 100 µL cell suspension was loaded into individual sample chambers and centrifuged in a Shandon CytoSpin centrifuge (Thermo Fisher Scientific, Wilmington, DE, US) at 800 rpm for 3 min. Immobilized cells were fixed (ICC-section) and subsequently subjected to immunocytochemistry, imaging and quantification. For each slide, three randomly picked areas (832 × 665.6 µM, 554 mM2) were selected for quantification. FITC stained cells and total number of cells was manually counted, and the proportion of FITC positive cells was calculated.
6
Murine Bronchoalveolar Lavage Analysis
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Bronchoalveolar lavage (BAL) fluid was collected from euthanized mice by lavaging the lung twice with 1 mL Hanks Balanced Saline Solution (21‐622CV, Corning) without Ca2+ and Mg2+. BAL fluid samples were centrifuged at 1500 × g for 10 minutes at 4°C, and the cell pellets were resuspended in 250 µL of PBS. Total cell counts were determined using a TC‐10 automated cell counter (Bio‐Rad), applied to slides using a Shandon Cytospin centrifuge (Thermo Scientific), and stained with Wright–Giemsa (89013, Thermo Scientific). Cell differentials were determined at 40× by a blinded observer counting at least 300 cells per sample.
7
Quantifying Inflammatory Cells in Guinea Pig Lungs
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After the final histamine challenge of the ovalbumin experiments or 24 h after the second LPS exposure, guinea-pigs were euthetized with an overdose of sodium pentobarbitone (Euthetal, 400 mg/kg i.p.). The trachea was cannulated and the lungs removed before instilling the left lung with saline (1 ml per 100 g body weight) for 3 min before withdrawal. This was repeated and the samples pooled for counting the total number of cells/ml with a Neubauer haemocytometer (Sigma-Aldrich, Gillingham, Dorset, UK). Differential counts of eosinophils, macrophages, lymphocytes and neutrophils were performed after centrifuging 100 µl of lavage fluid onto a glass microscope slide using a Shandon Cytospin centrifuge (ThermoFisher Scientific, Hemel Hempstead, UK) at 110 x g for 7 min.
Slides were then stained with 1.5% Leishman's solution in 100% methanol for 6 min. A minimum of 200 cells were counted.
Slides were then stained with 1.5% Leishman's solution in 100% methanol for 6 min. A minimum of 200 cells were counted.
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