Uptake of apoptotic cells by peritoneal MΦres was assessed as previously described (34 (link)). Briefly, thymocytes were collected from naive animals by mincing thymi through 2 μm gauze until completely homogenized. Erythrocytes were removed by incubating with red blood cell lysis buffer (Sigma-Aldrich). Thymocytes were resuspended at 1x10^7 cells/mL in complete DMEM and incubated in the presence of 0.1 µM dexamethasone (Sigma-Aldrich) at 37°C for 18 h. This produced >90% apoptosis, as assessed by Viastain AO/PI staining measured on a Cellometer ® Auto 2000 Cell Counter (Nexcelom Bioscience). Subsequently, apoptotic thymocytes were washed twice with PBS and resuspended in PBS at 10^6 cells/mL containing 40 ng/mL pHrodo-SE (Thermo Fisher Scientific) and incubated at RT for 30 min. Thereafter the cells were washed twice with PBS and resuspended in RPMI containing 5% foetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Unstained apoptotic cells served as staining control.
Cellometer auto 2000 cell counter
Manufactured by Revvity
Sourced in United States
The Cellometer Auto 2000 Cell Counter is an automated device designed to count and analyze cells. It provides accurate cell counts and viability measurements using fluorescence-based technology. The Cellometer Auto 2000 is capable of analyzing a wide range of cell types and sizes.
Automatically generated - may contain errors
Lab products found in correlation
Liberase tl, by Merck Group (5 mentions)
Dnase 1, by Merck Group (5 mentions)
Ammonium chloride potassium buffer, by Thermo Fisher Scientific (3 mentions)
Red blood cell lysis buffer, by Merck Group (2 mentions)
Protein transport inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Phrodo se, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Sr3677, by Bio-Techne (1 mentions)
Gsk 429286a, by Bio-Techne (1 mentions)
Tc s 7001, by Bio-Techne (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Chelex treated fetal calf serum, by Gemini Bio (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Easysep dead cell removal annexin 5 kit, by STEMCELL (1 mentions)
11 protocols using cellometer auto 2000 cell counter
1
Quantifying Phagocytosis of Apoptotic Cells
2
Cell Proliferation Assay for NHLFs and IPF Fibroblasts
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NHLFs and IPF fibroblasts (5 × 104) seeded on 24-well plates were grown overnight in serum-containing medium. On the following day, cells were switched to SFM supplemented with 0.1% BSA and treated with PDGF-BB (20 ng/ml) and different concentrations of IFN-γ and PFD for 1 or 3 days. At the end of the experiments, the cells were detached from the plate using BD cell detachment solution (Thermo-Fisher Scientific, Waltham, MA, USA). The cell number was counted by a Cellometer™ Auto 2000 cell counter (Nexcelom Bioscience, Lawrence, MA, USA). Cell viability was determined manually by counting cells after trypan blue staining using a Neubauer hematocytometer and an automated Cellometer.
3
Isolation and Culture of Primary Mouse Keratinocytes
4
Lung Tissue Dissociation and Cytokine Analysis
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Lung lobes were diced into small pieces and incubated in RPMI containing 0.33mg/mL Liberase TL and 0.1mg/mL DNase I (both from Sigma Aldrich) at 37°C for 45 minutes under agitation (200rpm). Enzymatic activity was stopped by adding FCS. Digested lung was filtered through a 70μm cell strainer and washed with RPMI. Red blood cells were lysed with the addition of ammonium-chloride-potassium buffer (Gibco) for 5 minutes at room temperature. Cells were then washed with RPMI supplemented with 10% FCS. Live cell numbers were enumerated using AOPI staining on a Cellometer Auto 2000 Cell Counter (Nexcelom).
For assessment of cytokine production, single cell suspensions were incubated in FCS-supplemented RPMI in the presence of 1X Protein Transport Inhibitor Cocktail (eBioscience) for 5 hours.
For assessment of cytokine production, single cell suspensions were incubated in FCS-supplemented RPMI in the presence of 1X Protein Transport Inhibitor Cocktail (eBioscience) for 5 hours.
5
Murine Lung Cell Isolation and Enumeration
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Lung lobes isolated from mice were washed with sterile 1× PBS, dissected into small pieces, digested in RPMI medium containing Liberase TL (0.33 mg ml−1; Sigma-Aldrich) and DNase I (0.1 mg ml−1; Sigma-Aldrich) at 37 °C for 45 min under agitation (200 r.p.m.) and added with FBS to block enzymatic digestion. Lung tissue was dispersed by passage through a 70-μm-pore-size cell strainer. Red blood cells were lysed with ACK buffer (Gibco) at room temperature for 3 min. Lung cells were washed with 1× PBS supplemented with 10% FBS, centrifuged at 1,500 r.p.m. for 5 min and the cell pellet resuspended in RPMI medium supplemented with 10% FBS. Cell numbers were counted using ViaStain acridine orange propidium iodide staining on a Cellometer Auto 2000 cell counter (Nexcelom).
6
Murine Lung Tissue Dissociation
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Lung lobes obtained from mice were washed with sterile 1× PBS, dissected into small pieces, and then digested in RPMI containing Liberase TL (0.33 mg/ml; Sigma-Aldrich) and DNase I (0.1 mg/ml; Sigma-Aldrich) at 37°C for 45 min under agitation (200 rpm). Enzymatic digestion was stopped by adding FBS. The digested lung tissue was dispersed by passage through a 70-μm pore-size cell strainer. Red blood cells were depleted with ACK lysing buffer (Gibco) at room temperature for 3 min. Cells were washed with 1× PBS supplemented with 10% FBS, centrifuged at 1,500 rpm for 5 min, and the cell pellet was resuspended in RPMI supplemented with 10% FBS. Live cell numbers were enumerated using ViaStain acridine orange propidium iodide staining on a Cellometer Auto 2,000 Cell Counter (Nexcelom). Single-cell suspensions were seeded on a round bottom 96-well plate for further analysis.
7
Lung Dissociation for Single-Cell Analysis
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Lung lobes were diced into small pieces and incubated in RPMI containing 0.33mg/mL Liberase TL and 0.1mg/mL DNase I (both from Sigma Aldrich) at 37°C for 45 minutes under agitation (150rpm). Enzymatic activity was stopped by adding FCS. Digested lung was filtered through a 70μm cell strainer and washed with RPMI. Red blood cells were lysed with the addition of ammonium-chloride-potassium buffer (Gibco) for 3 minutes at room temperature. Cells were then washed with RPMI supplemented with 10% FCS. Live cell numbers were enumerated using AOPI staining on a Cellometer Auto 2000 Cell Counter (Nexcelom).
8
Isolation and Enumeration of Peritoneal Cavity Exudate Cells
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Peritoneal cavity exudate cells (PEC) were obtained by washing the cavity with 10 mL lavage media comprised of RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 2 mM EDTA and 1% L-Glutamine (Thermo Fisher Scientific, Waltham, MA). Erythrocytes were removed by incubating with red blood cell lysis buffer (Sigma-Aldrich). Cellular content was assessed by cell counting using Viastain AO/PI solution on a Cellometer ® Auto 2000 Cell Counter (Nexcelom Bioscience, Manchester, UK) in combination with multicolour flow cytometry.
9
Isolation of Murine Lung Immune Cells
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Lung lobes were diced into small pieces and incubated in RPMI containing 0.33 mg/ml Liberase TL and 0.1 mg/ml DNase I (both from Sigma-Aldrich) at 37°C for 45 min under agitation (200 rpm). Enzymatic activity was stopped by adding FCS. Digested lung was filtered through a 70-μm cell strainer and washed with RPMI. Red blood cells were lysed with the addition of ammonium-chloride-potassium buffer (Gibco) for 5 min at room temperature. Cells were then washed with RPMI supplemented with 10% FCS. Live cell numbers were enumerated using ViaStain acridine orange propidium iodide staining on a Cellometer Auto 2000 Cell Counter (Nexcelom). For assessment of cytokine production, single-cell suspensions were incubated in FCS-supplemented RPMI in the presence of 1× Protein Transport Inhibitor Cocktail (eBioscience) for 5 h.
10
Cell Proliferation Assay of BM-MSCs
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Cell proliferation assays were performed on BM-MSCs and CBX-treated BM-MSCs. Cells were plated in 6-well plates at 1 × 105 cells/well. The number of cells at days 0, 1 and 2 were counted using Cellometer Auto 2000 Cell Counter (Nexcelom Bioscience, Waltham, MA, USA).
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