Valve cell phenotype was assessed at both the gene and protein level (see the Supplemental Experimental Procedures for additional details). Briefly, gene expression was measured by extracting RNA using a QIAGEN total RNA purification kit (QIAGEN) and reverse transcribed to cDNA using the SuperScript III RT-PCR kit with oligo(dT) primer (Invitrogen). Samples were amplified using the SYBR Green PCR system (Bio-Rad) on a Bio-Rad CFX96 cycler. For visualization of proteins, collagen constructs were fixed in 4% PFA overnight at 4°C. Cells were incubated overnight with the appropriate primary/secondary antibodies, washed, and nuclear counterstained for subsequent confocal imaging. For quantification of protein expression, cells were trypsinized from the collagen constructs, fixed with 4% PFA for 10 min, and then preserved in 50% methanol/PBS. Cells were further washed and incubated with appropriate primary/secondary antibodies and scanned using a Coulter Epics XL-MCL Flow Cytometer (Coulter). Adhesion testing assay methods are included in the Supplemental Experimental Procedures .
Coulter epics xl mcl flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Coulter Epics XL-MCL is a flow cytometer designed for laboratory applications. It is capable of analyzing and categorizing cells and particles in a fluid sample.
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Lab products found in correlation
Superscript 3 rt pcr kit with oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr system, by Bio-Rad (1 mentions)
Total rna purification kit, by Qiagen (1 mentions)
Cfx96 cycler, by Bio-Rad (1 mentions)
Cd11c pe, by BioLegend (1 mentions)
Expo32 adc software, by Beckman Coulter (1 mentions)
Cd45 pe cy5, by BioLegend (1 mentions)
Pe cy5 rat igg2b, by BioLegend (1 mentions)
Cd41 fitc, by BD (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Anti recoverin, by Merck Group (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apoptosis kit, by BD (1 mentions)
U0126, by Promega (1 mentions)
75 mm polypropylene tubes, by BD (1 mentions)
Act 5 diff, by Beckman Coulter (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Coulter dna prep reagents kit, by Beckman Coulter (1 mentions)
17 protocols using coulter epics xl mcl flow cytometer
1
Valve Cell Phenotype Characterization
2
Quantification of E-cadherin and Vimentin
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Flow cytometry for E-cadherin 1:100 (Abcam) and vimentin 1:100 expressing cells was performed. Briefly, cells were trypsinized, fixed with 4% PFA for 10 min and then preserved in 50% methanol/PBS. Cells were kept in the -20C until antibody staining was preformed. Samples were divided into multiple aliquots in order to stain the proteins separately and compensate for secondary antibody non-specific binding. Cells were incubated for 24 hrs at 4 C in primary antibody diluted in either PBS (extracellular) or 0.2% saponin-PBS (intracellular). Cells were then washed 3 times with PBS and incubated with appropriate secondary antibodies and imaged using a Coulter Epics XL-MCL Flow Cytometer (Coulter). All samples were compensated using appropriate background subtraction and all samples were normalized using 7500 cells per flow condition.
3
Flow Cytometry Technique for Murine Blood
4
Quantitative Flow Cytometry and HCV PCR
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Flow cytometry was carried out using a COULTER Epics XL MCL flow cytometer produced by the Beckman Coulter Company. Fluorescent-labeled D3/CD4/CD8 monoclonal antibodies and red blood cell lysing liquid were selected from original OptiClone reagents. The polymerase chain reaction (PCR) amplification was carried out using a LightCycler fluorogenic quantitative nucleic acid amplifier. The HCV nucleic acid quantification kit was selected from kits produced by the Shanghai Kehua Bio-engineering Co., Ltd.
5
Intracellular Calcium Dynamics in Rho P23H/+
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Intracellular calcium levels were determined with the calcium probe Fluo-4 AM (Life Technologies), as previously published [14, 23] . Briefly, wild type and Rho P23H/+ retinas at PN19 were harvested and incubated with 19 U/ml of papain for 30 min and, after 33-fold dilution with DMEM containing 10 U/ml DNAse, retina cells were dissociated by trituration.
After three washes with PBS, cells were incubated with 1 M Fluo-4 AM at 37°C for 30 min in Ca 2+ -free medium. Photoreceptor cells were characterized by staining with anti-Rho antibody 1D4 (1:1000, Sigma) and with anti-Recoverin (1:500, Millipore), as in [14, 23] , and plotted over the forward scatter to define the gating strategy for the following intracellular calcium analysis. Retinal cells from four wild type and four Rho P23H/+ mice, derived from at least three different litters, were gated. Fluorescence was measured in 20,000 events with a Coulter Epics XL-MCL flow cytometer (Beckman Coulter) at an excitation wavelength of 488 nm. Fluo-4 AM median fluorescence intensity (MFI) was measured in cells from each dissociated Rho P23H/+ retina and compared to MFI from wild type photoreceptors from littermates.
After three washes with PBS, cells were incubated with 1 M Fluo-4 AM at 37°C for 30 min in Ca 2+ -free medium. Photoreceptor cells were characterized by staining with anti-Rho antibody 1D4 (1:1000, Sigma) and with anti-Recoverin (1:500, Millipore), as in [14, 23] , and plotted over the forward scatter to define the gating strategy for the following intracellular calcium analysis. Retinal cells from four wild type and four Rho P23H/+ mice, derived from at least three different litters, were gated. Fluorescence was measured in 20,000 events with a Coulter Epics XL-MCL flow cytometer (Beckman Coulter) at an excitation wavelength of 488 nm. Fluo-4 AM median fluorescence intensity (MFI) was measured in cells from each dissociated Rho P23H/+ retina and compared to MFI from wild type photoreceptors from littermates.
6
Apoptosis and Cell Cycle Analysis
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Apoptosis and cell cycle analysis were assessed by flow cytometry as previously described.29 Briefly, 24 h after treatment with undiluted and 6.25% diluted extracts for 24 h, human gingival fibroblasts and keratinocytes were harvested, re‐suspended in 0.5 mL hypotonic propidium iodide (PI) solution (50 μg/mL propidium iodide in 0.1% sodium citrate plus 0.1% TritonX‐100) and analysed by flow cytometry using Coulter Epics XL‐MCL Flow Cytometer (BeckmanCoulter). Data were analysed using FlowJo software (TreeStar).
7
Radiation-Induced Apoptosis Assay
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Cells were treated with 5 Gy ionizing radiation (IR) and incubated with U0126 (Promega, WI, USA), AG1024, or LY294002 (Calbiochem, Darmstadt, Germany) at 37°C for 24hours, as described previously [21] (link). Apoptosis was assayed using an Annexin V Apoptosis Kit (BD Pharmingen, San Diego, CA) and analyzed using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA). Cells were considered to be apoptotic if they stained positive for both Annexin V and PI (Ann+/PI+). The proportion of IR-induced apoptotic cells was determined by subtracting the proportion of apoptotic cells detected in the absence of IR.
8
Apoptosis Quantification by Flow Cytometry
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Cell apoptosis was also assessed by flow cytometry after Annexin V-FITC/PI staining as manufacturer's instructions. MEFs were transfected with scramble NC (NC), miR-93 mimics (93), NC inhibitor (IN-NC), or miR-93 inhibitor (IN-93) individually for 12 h, followed by the treatment of hypoxia for another 24 h. The normal group was set as control. Analyses were carried out with a COULTER EPICS XL-MCL™ flow cytometer (Beckman Coulter) equipped with Expo32 ADC analysis software. The cellular apoptosis rate was mapped by GraphPad Prism 5.
9
Apoptosis Analysis of PASMCs
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At the end of all interventions, PASMCs (>1×106 cells) were collected and stained with Annexin V‐FITC/propidium iodide Kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. The apoptosis of PASMCs was measured by a Coulter Epics XL‐MCL™ Flow Cytometer (Beckman Coulter, Inc, Indianapolis, IN).
10
Cell Cycle Analysis by Flow Cytometry
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Cell cycle was analyzed by flow cytometry. One-hundred-thousand cells were seeded into six well plates and treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin. Twenty-four hours and 48 h later, the culture medium of each sample was collected and centrifuged (400× g, 7 min) in order to recover apoptotic cells. Attached cells were washed twice with PBS and resuspended with 0.5 ml of hypotonic propidium iodide solution (50 μg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) in 12 × 75 mm polypropylene tubes (BD Biosciences). The tubes were kept at 4°C for at least 30 min before flow cytometry analysis. Flow cytometry experiments were performed using Coulter Epics XL-MCL Flow Cytometer (Beckman Coulter) and data were analyzed using FlowJo software (TreeStar).
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