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11 protocols using isoflow sheath fluid

1

Quantification of NIS and PENDRIN Proteins

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5 × 106 leukocytes were washed in PBS and then incubated in 500 µL PBS with 10% goat serum (S-1000, Vector Laboratories) for 30 min to block non-specific binding. The cells were resuspended in 100 µL PBS with 10% goat serum for 1 h at room temperature with primary or without (control) rabbit antibodies against SLC5A5/NIS (SAB2102220, Sigma) or SLC26A4/PENDRIN (MBS9215961, MyBioSource). The cells were washed and then stained for 30 min with secondary F(ab′)2 goat anti-rabbit (Invitrogen, A21246) at a 1:100 dilution and CD45 Krome orange (Beckman Coulter, A96416) at a 1:20 dilution at room temperature. Other experiments included CD14 FITC staining (BD Pharmingen, 555397) to identify monocytes. Cells were washed, resuspended in IsoFlow sheath fluid (Beckman Coulter), and then loaded onto BD FACSCanto II where 25,000 events were collected in the lymphocyte gate. The resulting data and the median fluorescence intensity (MFI) were analyzed utilizing FlowJo software.
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2

Profiling T Cell Subsets and Tfh

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To determine the percentage of different T cell subsets, at least 1 × 106 PBMCs were suspended in isoflow sheath fluid (Beckman Coulter) and stained in a DuraClone IM T cell tube (Beckman Coulter, Miami, FL) according to the manufacturer's protocol. The T cell tube contained anti-CD45, CD3, CD8, CD4, CD45RA, and CCR7. Samples were measured by use of the Navios flow-cytometer (Beckman Coulter).
To determine the Tfh, at least 1 × 106 PBMC were washed by Fascflow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark.
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3

Mutagenesis and Screening of C. cellulolyticum DPEase

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A library of DPEase of C. cellulolyticum mutants was generated following the error-prone PCR protocol (35 ) using the OneTaq DNA Polymerase (New England Biolabs), the forward primer 5′-GCCGTCTCGGATGAAACACGGTATCTACTAC-3′, the reverse primer 5′-GCCGTCTCCCGCTTTAAGAGTGTTTGTGGCATTC-3′ and as template a gBlock encoding the C. cellulolyticum DPEase. A control library was performed with the Q5® High-Fidelity DNA Polymerase (New England Biolabs). Each library was inserted in the Mutant Drop Zone (MDZ) downstream of the psicose biosensor (BBa_K2448057) by Golden Gate, using the BsmBI restriction enzyme (Thermo Fisher Scientific). Ten microliters of the Golden Gate reaction were used to transform chemically competent E. coli DH5α cells. After overnight culturing in LB media supplemented with 35 µg/ml chloramphenicol, transformed cells were centrifuged, washed with IsoFlow Sheath Fluid (Beckman Coulter) and resuspended in this same isotonic fluid at a concentration of 106 cells/ml. Flow cytometric measurements were performed at Genoscope on a MoFlo Astrios cell sorter (Beckam Coulter), using a single laser operating at 488 nm for excitation and a channel of 576/21 nm for detection of the mEmerald fluorescence. The selection was triggered by fluorescence (threshold 0.05%). The data were analyzed using the Summit V6.2 Software (Beckam Coulter).
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4

Apoptosis Assay in HCEnC-21T Cells

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HCEnC-21T cells were seeded in a 6-well plate and cultured for 24 h. Cells were treated with/without MN (50 μM) or 4-OHE2 (10 μM), and incubated for 16 h in estrogen-free medium. Cells were then trypsinized, washed in cold PBS, and incubated in the dark with 3 μL of Annexin-V/FITC (Ann) (Thermo Fisher Scientific) and 2 μL of propidium iodide (PI, Sigma-Aldrich) in a total volume of 50 μL for 30 min at room temperature. Cells were re-suspended in 2 mL of Isoflow sheath fluid (Beckman Coulter, Fullerton, CA) and 20,000 live cells were acquired on a LSR II (BD Biosciences) with DIVA software (BD Biosciences) gated for PI and Annexin V with unstained cells as a negative control. Data were then analyzed with Summit 4.3 (Beckman Coulter, Fullerton, CA).
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5

Phenotypic Characterization of Stem Cells

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Flow cytometry was used to characterize CD90 and CD105 cell surface markers using CD90-FITC Mouse IgG (IM1839U) and CD105-PE Mouse IgG (A07414, Beckman Coulter). Additionally, CD34 (IM1870) and CD45 (A07782, Beckman Coulter) were used for negative selection. Fluorochrome-conjugated antibodies were incubated in 20 μL/2.0 × 105 cell suspension in full media or PBS for 20 minutes at room temperature protected from light. Finally the cells were washed three times with PBS solution, resuspended into IsoFlow Sheath Fluid (8546859, Beckman Coulter), and the cell fluorescence was measured using Beckman Coulter Cytomics FC500 instrument. The data was assessed with Kaluza analysis software. Positivity for each antibody was defined as the level of fluorescence >99% of the isotype-matched control antibodies.
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6

MN-Induced Apoptosis in NQO1 Knockout Cells

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HCEnC-21T control (NQO1+/+) and NQO1/ cells were seeded in a 6-well plate and cultured for 24 hours prior to treating with 50μM MN in low-glucose DMEM) and incubated for 5 hours. Cells were then trypsinized, washed in cold PBS, and incubated in the dark with 3μl of Annexin-V/FITC (Ann) (Thermo Fisher, Waltham, MA) and 2μl of PI (Sigma-Aldrich) in a total volume of 50μl for 30 minutes at room temperature. Cells were re-suspended in 2ml of Isoflow sheath fluid (Beckman Coulter, Fullerton, CA) and 20,000 live cells were acquired on a LSR II (BD Biosciences) with DIVA software (BD Biosciences) gated for PI and Annexin V with unstained cells as negative control. Data were then analyzed with Summit 4.3 (Beckman Coulter, Fullerton, CA).
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7

Flow Cytometric Analysis of Immune Complexes

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Immune complexes were analyzed on the flow cytometer, Cytomics FCM 500 from Beckman Coulter 22, Avenue des Nations CS 54359, 93420, Villepinte. It is equipped with 2 lasers 488 nm, 40 mW and 638 nm, 25 mW. It has the ability to analyze events on five colors: 525 nm, 575 nm, 620 nm, 675/695 nm, and 755 nm.
The sheath fluid for the cytometer was IsoFlow™ Sheath Fluid from Beckman Coulter, Part Number 8448010, an isotonic fluid at a pH 7.35-7.65, with Sodium Phosphate Dibasic, Sodium Fluoride, Diethylene Glycol Phenyl Ether, and 2-Phenoxyethanol.
A microparticle gate was established in agreement with the published method for characterization of circulating cell-derived microparticles by preliminary standardization experiments using a blend of size-calibrated 0.3, 0.5, 0.9, and 3 μm fluorescent beads, Megamix®, Biocytex, Marseille, France [27 (link), 28 (link)]. To enlarge the scale of fluorescent beads, in some experiments, Trucount beads (Beckton Dickinson) of 10 μm were added.
A gate for DNA-anti-DNA immune complexes for each series of experiments was built around DNA-anti-DNA immune complexes observed in the aliquot of the pool of 15 SLE serum samples incubated with calf thymus DNA. See below for more details and for compensations.
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8

Erythrocyte Membrane Particles Analysis

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EryMPs were analyzed in flow cytometer FC500 (Beckman Coulter, Brea, CA, USA), using IsoFlow Sheath Fluid (Beckman Coulter) as fluid phase. The laser was set at 488 nm, thresholds for forward scatter (FS) and side scatter (SS) were set to 2. Fluorescence channels, FS and SS were set at logarithmic gain. The flow cytometry data were analyzed using FlowJo 8.7.1 (Tree Star, Inc., Ashland, OR, USA). Data was collected with live gate on erythrocyte surface marker glycophorin A (anti-CD235a-PE detected in FL2).
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9

Automated Algal Cell Size Measurement

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The cell size was measured with a Beckman Coulter Multisizer 4e analyzer using 0.2 mm filtered isotonic II (Beckman Coulter) as the diluent and blank. The samples were fixed with 0.5% glutaraldehyde and were diluted 10,000 times with filtered IsoFlow Sheath Fluid (Beckman Coulter), and 500 μL of algal culture was analyzed each time. Particle size distribution was obtained with a 70 μm aperture, which measured particle size ranging from 1.4 to 56 μm. Data on cell diameter and density of a peak were obtained with the software Multisizer v4.03 (Beckman Coulter). Raw data sets were retrieved for plotting size distribution patterns.
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10

Simultaneous Apoptosis and GD2 Staining

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Enriched CD24-positive cells (~107) were simultaneously stained with Vybrant™ FLICA pan-Caspase Apoptosis Assay Kits for flow cytometry (Invitrogen/Thermo Scientific, Waltham, MA, USA) 1:150 and APC conjugated anti-human Ganglioside GD2 Antibody (BioLegend, San Diego, CA, USA) 1:100 in 0.25 mL of IsoFlow Sheath Fluid (Beckman Coulter, Pasadena, CA, USA) for 30 min at 37 °C. Then the cells were washed once with warm IsoFlow fluid and measured by a Sony SH800Z Sorting Flow Cytometer (Sony Biotechnology, Tokyo, Japan) with automatic channel compensation.
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