Cells were trypsinized and analyzed for GFP signal using a standard FACScan cytometer (BD Biosciences, San Jose, CA, USA), as we have described.16 (link) Briefly, cells were fixed with 0.4% paraformaldehyde (PFA) before treating with anti-myosin heavy chain antibody (Developmental Studies Hybridoma Bank, Iowa city, IA, USA) in PBS with 5% donkey serum and 20 µg/mL DNAse-free RNAse (Sigma-Aldrich Co., St Louis, MO, USA), overnight at 4°C. Cells were also stained with 40 µg/mL 7-aminoactinomycin D (7AAD, BD Biosciences) to measure the DNA content. Data were collected using a standard FACScan cytometer (BD Biosciences) upgraded to a dual laser system with the addition of a blue laser (15 mW at 488 nm) and a red laser (25 mW at 637 nm Cytek Development, Inc., Fremont, CA, USA). Data were acquired using CellQuest software (BD Biosciences) and analyzed using FlowJo software (Ver9.4 Treestar Inc., San Carlos, CA, USA). Cells stained with isotype-matched IgG antibodies were used as controls to determine the positive cell population.
Facscan cytometer
Manufactured by BD
Sourced in United States
The FACScan cytometer is a flow cytometry instrument designed for automated cell analysis. It can rapidly measure and analyze multiple physical and fluorescent characteristics of individual particles or cells within a fluid sample. The FACScan cytometer is capable of detecting and quantifying a wide range of cell types and analytes.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (25 mentions)
Propidium iodide, by Merck Group (7 mentions)
Facscalibur, by BD (4 mentions)
Cellquest pro software, by BD (4 mentions)
Dnase, by Merck Group (3 mentions)
Nonidet p 40, by Merck Group (3 mentions)
Sodium citrate, by Merck Group (3 mentions)
Rnase a, by Merck Group (3 mentions)
Cellquest, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cellquest analysis software, by BD (2 mentions)
Luciferase assay system, by Promega (2 mentions)
Perc cy5 anti f4 80, by BD (2 mentions)
Anti ly6g pe cy7, by BD (2 mentions)
Pe cy3 anti p stat6, by BD (2 mentions)
Bv421 anti siglec f, by BD (2 mentions)
Anti cd11b apc cy7, by BD (2 mentions)
Anti cd206 apc, by BioLegend (2 mentions)
Gemcitabine, by Merck Group (2 mentions)
Apc conjugated anti cd206, by BioLegend (2 mentions)
181 protocols using facscan cytometer
1
Flow Cytometric Analysis of Myosin Heavy Chain
2
Assessing Stem Cell Phenotype and Apoptosis
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For the stemness assay, cells with an anti–CD44‐PE/Cy7 antibody (BioLegend, cat. no. 372810) and submitted for analysis on a FACScan cytometer using FlowJo software (version 10; BD Biosciences). For the apoptosis assay, cells were stained with propidium iodide and FITC‐conjugated annexin V and analyzed on a FACScan cytometer using FlowJo software (version 10; BD Biosciences). Each experiment was performed in triplicate.
3
Docetaxel-Induced Apoptosis Quantification
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1 × 106 cells were seeded in 100-mm plates and after 24 h treated with 10 nmol l−1 docetaxel for 12 h and 24 h. After the treatment, the cells were trypsinized, washed with cold PBS, and centrifuged at 700g for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 ml of cold PBS. The cells were counted with a Neubauer chamber, and 1 × 105 cells were transferred to a cytometry tube. Then, the cells were processed and labeled according to the BD Pharmingen Annexin V-FITC Apoptosis Detection Kit instructions (Cat. No. 556547, Becton Dickinson, Franklin Lakes, NJ, USA). Finally, labeled cells were analyzed using a FACScan cytometer (Becton Dickinson), and the analysis of the results was made using the FCS Express Plus software (DeNovo Software, Glendale, CA, USA).
4
Cell Cycle Analysis of BGC-823 Cells
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BGC-823 cells (1×106) were seeded in 6-well plates for 24 h. After induction with HPRP-A2 (5, 10, 15 μM) for 24 h, cell cycle distribution was determined by a FACScan cytometer and Cell Quest software (FACS Calibur, Becton-Dickinson, San Jose, CA, USA). All experiments were performed in triplicates.
5
Quantifying Monocyte Phagocytosis of Fungal Pathogens
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Monocytes were pre-treated or not with MBCD (5 mM) or DAmb (5 μg/ml) for 30 minutes at 37°C and then incubated for 1h at 37°C with FITC-conjugated heat killed or live C. albicans, Staphylococcus aureus at a ratio of 1:5 or with FITC-conjugated β-glucan (25 μg/ml) in RPMI+1% heat inactivated FBS.
In some experiments monocytes were pre-treated for 30 minutes at 37°C with laminarin, mannan, GlcNAc (100 μg/ml each) or with a purified mouse blocking mAb anti-human CD11b/Mac-1 (clone ICRF44, 50 μg/ml) before fungal stimulation.
The percentage of phagocytosing cells were examined on a FACScan cytometer equipped with Cellquest Software (Becton Dickinson) and determined by comparing the intensity of FITC before and after trypan blue (Sigma) quenching of membrane-bound microrganisms or β-glucan, leaving unchanged the signal coming from the engulfed ones. As a control, cells incubated with the microorganisms at 0°C were used to exclude background staining.
In some experiments monocytes were pre-treated for 30 minutes at 37°C with laminarin, mannan, GlcNAc (100 μg/ml each) or with a purified mouse blocking mAb anti-human CD11b/Mac-1 (clone ICRF44, 50 μg/ml) before fungal stimulation.
The percentage of phagocytosing cells were examined on a FACScan cytometer equipped with Cellquest Software (Becton Dickinson) and determined by comparing the intensity of FITC before and after trypan blue (Sigma) quenching of membrane-bound microrganisms or β-glucan, leaving unchanged the signal coming from the engulfed ones. As a control, cells incubated with the microorganisms at 0°C were used to exclude background staining.
6
Immunophenotypic Analysis of Cells
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Phenotypic analysis of cells was carried out by direct or indirect immunofluorescence staining, depending on whether or not the primary mAb was fluorochrome-conjugated or unconjugated, respectively, on a FACScan cytometer (Becton-Dickinson) as previously described [10] (link). Cytofluorometer data were analysed using the CellQuest program (Becton-Dickinson). A minimum of 4000 events per sample was analyzed.
7
Apoptosis Evaluation via PI Staining
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The percentage of the apoptotic cells was evaluated by propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA) staining and flow cytometry at 24, 48 or 72 h from the administration of sodium mesoglycan and Prisma® Skin. The analysis was performed with FACScan cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) by Cell Quest program, version 6.0.
8
Lentiviral Vector Production and Titration
9
Endocannabinoid-Induced Apoptosis Quantification
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Cells were grown at a 106 density and treated with endocannabinoids at a 5.0 μM concentration for 6 h. After the treatment, the cells were trypsinized, washed with cold PBS and then centrifuged at 2,500 rpm for 5 min. The supernatant was discarded and the pellet was resuspended in 1 ml of cold PBS. The cells were counted with a Neubauer chamber and 105 cells were transferred to a cytometry tube. Then, the cells were processed and labeled according to the BD Pharmingen Annexin V-FITC Apoptosis Detection kit that was used for this assay. The labeled cells were analyzed in a FACScan cytometer (Becton-Dickinson). Data analysis was assessed using WinMD1 version 2.8 software.
10
Cell Cycle Analysis by Flow Cytometry
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Cells were incubated with D2O as indicated for 24 hrs. Nocodazole (1µg/mL, applied for 2 hrs) was used as positive control to induce G2/M arrest. After fixation in ice-cold 70% ethanol cells were incubated in PBS containing 40g/mL RNase A for 30 mins at 37°C and resuspended in PBS containing 50g/mL propidium iodide. Analysis of cell cycle was assessed by a FACScan Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as described.13 (link)
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