dTHP1 cells that were 80% confluent were treated with Fn for 24 h. Cells were observed under an inverted microscope (Nikon TE 300), harvested in 5 mM EDTA in PBS and washed. For analysis of CCR7, CD206, and HLA-DR, cells were resuspended with 300 µl of PBS and 5 µl of PE-conjugated anti-human CCR7 and CD206 (eBioscience) or FITC-conjugated anti-human leukocyte antigen (HLA)-DR (BD Biosciences, Erembodegem, Belgium) for 15 min and then analyzed by a FACS Calibur (Beckman-Coulter, Miami, USA). For analysis of cell apoptosis, cells were resuspended with annexin-binding buffer and then stained with annexin V and propidium iodide (PI) according to the manufacturer’s instructions (BD Biosciences). Apoptotic cells were analyzed by a FACS Calibur (Beckman-Coulter).
Facscalibur
Manufactured by Beckman Coulter
Sourced in United States, China
The FACSCalibur is a flow cytometry instrument designed for multiparameter analysis of cells and particles. It utilizes a combination of laser excitation and detectors to provide information on the size, granularity, and fluorescence characteristics of individual cells or particles within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (5 mentions)
Rnase a, by Merck Group (3 mentions)
Software, by FlowJo (3 mentions)
Rnase, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Euroclone (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Propidium iodide, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by BD (2 mentions)
Epics xl mcl, by Beckman Coulter (1 mentions)
Act5 diff cp, by Beckman Coulter (1 mentions)
Methylcellulose medium, by STEMCELL (1 mentions)
Mouse lineage cell depletion kit, by Miltenyi Biotec (1 mentions)
Mil 3, by Thermo Fisher Scientific (1 mentions)
Human il 6, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Multicycle av dna analysis software, by Phoenix Pharmaceuticals (1 mentions)
Cxcr5, by BioLegend (1 mentions)
Cd138, by BioLegend (1 mentions)
Kaluza analysis 2, by Beckman Coulter (1 mentions)
86 protocols using facscalibur
1
Evaluating Macrophage Immune Markers
2
Quantifying LDLR Expression and Internalization
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Flow cytometry was used to detect the amount of cell-surface LDLR expression and LDL internalization. The fluorescence of 10,000 events for each sample was acquired for data analysis. Forward scatter versus side scatter gates were set to exclude dead cells and debris. Experiments were repeated at least three times with triplicate samples for each cell line.
To measure the amounts of cell-surface LDLR expression, the cells transfected with wild-type, G615V or C201F LDLR were harvested 48 hours after transfection and resuspended in PBS containing 1% BSA. Then, the cells were washed twice in PBS containing 1% BSA and incubated with phycoerythrin (PE)-conjugated mouse monoclonal anti-human LDLR (1∶20, R&D) at room temperature for 30 minutes in the dark. Finally, the cells were washed three times with PBS containing 0.1% BSA and resuspended in PBS. PE fluorescence was quantified using a FACS Calibur (Beckman Coulter).
To measure LDLR internalization activity, the cells transfected with wild-type, G615V or C201F LDLR were incubated in serum-free media containing 20 μg/ml Dil-LDL for 4 hours at 37°C. Following the LDL incubation, the medium was removed, and the cells were detached from the culture dish. Afterwards, the cells were washed twice in PBS and resuspended in PBS. The amount of LDL internalization was detected using a FACS Calibur (Beckman Coulter).
To measure the amounts of cell-surface LDLR expression, the cells transfected with wild-type, G615V or C201F LDLR were harvested 48 hours after transfection and resuspended in PBS containing 1% BSA. Then, the cells were washed twice in PBS containing 1% BSA and incubated with phycoerythrin (PE)-conjugated mouse monoclonal anti-human LDLR (1∶20, R&D) at room temperature for 30 minutes in the dark. Finally, the cells were washed three times with PBS containing 0.1% BSA and resuspended in PBS. PE fluorescence was quantified using a FACS Calibur (Beckman Coulter).
To measure LDLR internalization activity, the cells transfected with wild-type, G615V or C201F LDLR were incubated in serum-free media containing 20 μg/ml Dil-LDL for 4 hours at 37°C. Following the LDL incubation, the medium was removed, and the cells were detached from the culture dish. Afterwards, the cells were washed twice in PBS and resuspended in PBS. The amount of LDL internalization was detected using a FACS Calibur (Beckman Coulter).
3
Cell Cycle Analysis of Thymocytes
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Thymocytes were harvested and fixed in ice-cold 70% ethanol. On staining, cells were suspended in PBS containing 50 mg/ml RNase A (Sigma) for 15 min at room temperature. Last, we added propidium iodide (Sigma) staining solution at a final concentration of 50 µg/ml. cell cycle profiles were generated on a FACS-Calibur and Coulter Epics XL-MCL flow cytometer. The experiment was repeated 3 times for each cell type and condition.
4
Blood Cell Characterization in HIV
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Full blood count and CD4+ cell counts were done using the Coulter ACT 5Diff CP and Facscalibur, as per standard operating procedures at the ISS clinic. Reference ranges as determined for the HIV population were used.8 (link) Thrombocytopenia was regarded as platelet count less than 150×109/L, anemia was defined as hemoglobin concentration less than 12 g/L for adult females and less than 13 g/dL for adult males, and leucopenia was considered as white blood cell (WBC) count less than 2.75×109/L. Thin blood films were prepared, stained by Giemsa and examined for anemia typing based on: a) size of red blood cells (normocytic, microcytic and macrocytic) and b) hemoglobin content (normochromic and hypochromic). Pancytopenia was considered if all the three blood cells were below their minimum for a reference range. We ensured strict adherence to the standard operating procedures, the hematology analyzer was well maintained and controls were run daily before use. A hematology bench aid was used to ascertain cell features of a comprehensive film comment.
5
Annexin V-FITC Apoptosis Assay for HCT-116 Cells
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The Annexin fluorescein isothiocyanate (V-FITC) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to measure HCT-116 cell apoptosis according to the manufacturer’s protocol. Briefly, the HCT-116 cells were seeded in complete culture medium and incubated with EGLP (150 and 300 µg/ml) for 48 h. Then, the HCT-116 cells were collected by centrifugation at 10,000 rpm for 5 min at 4°C and resuspended in 500 µl Annexin V-FITC binding buffer. HCT-116 cells were incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) in the dark for 15 min. The apoptosis rates were quantitatively analyzed with a FACSCalibur flow cytometer (Beckman Coulter, Brea, CA, USA).
6
Murine Hematopoietic Stem Cell Assay
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Single‐cell suspensions from BM and PB were stained with panels of fluorochrome‐conjugated antibodies (Table S1 ). Flow cytometric analysis of HSPCs was performed as previously described (Cao, Cai, et al., 2015 ). The analyses were performed using a FACSCalibur or Beckman Coulter Gallios. All data were analyzed using FlowJo software or Kaluza. BM lin− cells were sorted using Mouse Lineage Cell Depletion Kit (130090858, Miltenyi Biotec). Cells were sorted by BD FACSAria III. LSK cells were plated in triplicate in methylcellulose medium (03134, STEMCELL Technologies) supplemented with mouse stem cell factor (mSCF) (100 ng/ml), mouse IL‐3 (mIL‐3) (10 ng/ml), mouse erythropoietin (mEPO) (4 U/ml), mouse thrombopoietin (mTPO) (50 ng/ml), mouse granulocyte–macrophage CSF (mGM‐CSF) (10 ng/ml), and human IL‐6 (50 ng/ml, PeproTech). The cells were then scored on day 10 of the cultures at 37°C and 5% CO2.
7
Cell Cycle Analysis of PC9 Cells
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This procedure was reported previously [32 (link)]. In brief, PC9 cells were cultured in 6-well culture plates at 3 × 105 cells/well and treated with increased doses of PPI for 24 h. Afterwards, the cells were washed and resuspended in PBS and incubated with 0.1 % sodium citrate containing propidium iodide (PI) 0.05 mg and 50 μg RNase for 1 h at room temperature (RT). The cells were washed and subjected to FACScalibur flow cytometric analysis (FC500, Beckman Coulter, FL, USA), and the proportion of cells within the G0/G1, S, and G2/M phases of the cell cycle were analyzed using the MultiCycle AV DNA Analysis software (Phoenix Flow Systems, Inc. San Diego, CA, USA).
8
Comprehensive Lymphoid Cell Phenotyping
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Thymus, spleen and lymph node cell suspensions were prepared; erythrocytes were lysed, and cells were counted. Cell samples were stained with combinations of fluorescently labelled antibodies to the cell surface markers CD19, CD3, CD4, CD8, CD43, CD25, Gr1, CD11b, B220, F4/80, IgD, CD21, CD23, CD69, CD86, CD95, GL7, PD-1 (all from BD Biosciences), CD138, CXCR5 (both from Biolegend) and IgM (Jackson Immunoresearch Lab.) for 30 min at 4°C. Cell analysis was performed in a FACScalibur, Beckman Coulter CYTOMIX FC500 MCL and LSR-II cytometer (BD Biosciences). Biotinylated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 antibodies (Southern Biotech) were used to detect surface expression of IgG isotypes, followed by fluorescently labelled streptavidin (Molecular Probes). The profiles obtained were analysed with FlowJo (BD Biosciences) and Kaluza Analysis 2.11 (Beckman Coulter) software; B cells were gated as CD19+ cells when indicated.
9
Cell Cycle Analysis of Transfected Cells
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Transfected ES-2 or A2780 cells were seeded into 6-well plates for 24h in complete medium before cells were deprived of serum for 48h and then returned to complete medium for an additional 24h. All cells were collected by centrifugation, fixed in 95% ethanol, incubated at -20°C overnight and washed with phosphate buffered saline (PBS). Then, cells were resuspended in 1ml FACS solution (PBS, 0.1% TritonX-100, 60 ug/ml propidium iodide (PI), 0.1 mg/ml DNase free RNase, and 0.1% trisodium citrate). After a final incubation on ice for 30 min, cells were analyzed using a FACS Calibur flow cytometer (Beckman Coulter). A total of 10,000 events were counted for each sample.
10
Cell Cycle and Apoptosis Analysis
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After 48 h of transfection with siRNA, the cell pellet was collected and washed twice with cold PBS. The cells were fixed with cold 70% ethanol at room temperature overnight. The supernatant was removed by centrifugation the next day and washed with PBS again. The cells were then stained with propidium iodide (PI; BD Biosciences). To detect apoptosis, the cell pellet was collected, washed twice with cold PBS, combined with Annexin V‐FITC and PI, and was stained for 15 min at room temperature. Finally, an FACS Calibur flow cytometer (Beckman Coulter) was used to analyze the cell cycle and apoptosis. The cell cycle results were analyzed using Kaluza v2.1.1 software.
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