Antibody-dependent neutrophil-mediated phagocytosis was assessed by the measurement of the uptake of antibody-opsonized, antigen-coated fluorescent beads by primary neutrophils. Biotinylated Env gp120 was used to saturate the binding sites on 1 pm fluorescent neutravidin beads (Invitrogen). Excess antigen was removed by washing the 28 beads, which were then incubated with patient Ab samples for 20 minutes at 37°C. Leukocytes were isolated from blood collected from HIV-seronegative donors by ACK lysis of red blood cells. Following opsonization, the freshly isolated leukocytes were added, and the cells were incubated for 1 hr at 37°C to allow phagocytosis. The cells were then stained for CD66b to identify neutrophils, and fixed, and the extent of phagocytosis was measured via flow cytometry on a Stratedigm S1000EXi flow cytometer equipped with a high-throughput sampler. The data are reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis (integrated MFI: frequency x MFI) (Darrah et al., 2007 (link)).
S1000exi flow cytometer
Manufactured by Stratedigm
Sourced in United States
The S1000EXi Flow Cytometer is a high-performance instrument used for the analysis and sorting of cells and particles. It is designed to precisely detect and measure various cellular characteristics, such as size, granularity, and fluorescence signals, through the application of flow cytometry technology.
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Lab products found in correlation
Cellrox deep red reagent, by Thermo Fisher Scientific (3 mentions)
Cd4 pe cy7, by Thermo Fisher Scientific (2 mentions)
Ifn γ fitc, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mitotracker green fm dye, by Thermo Fisher Scientific (2 mentions)
Fluorescent neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
Rpmi 1640, by Welgene (1 mentions)
Apc il 17, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis buffer, by BD (1 mentions)
Vegfr2 apc, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Penicillin streptomycin solution, by Sartorius (1 mentions)
20 protocols using s1000exi flow cytometer
1
Antibody-Mediated Neutrophil Phagocytosis Assay
2
Flow Cytometric Analysis of Fluorescent Reporters
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Ten thousand events per sample were analysed with a Stratedigm S1000EXI flow cytometer (Stratedigm, California, USA) using 405 nm excitation with emission collected between 565 and 595 nm (for Zombie Yellow), 405 and 552 nm excitation with emission collected between 415 and 475 nm (for mTurquoise), 488 nm excitation with emission collected between 515 and 545 nm (for Alexa Fluor 488/BDP-FL) and 642 nm excitation with emission collected between 661 and 690 nm (for Cy5/SNAP-Cell SiR 647). FCS3.0 files were exported using CellCapTure Analysis Software (version 4.0 RC4, Stratedigm, California, USA) and imported into FlowJo (version 8, Tree Star, Oregon, USA).
Following gating (Supplementary Figs.15 and 16 ), the geometric mean fluorescence intensity of untreated cells was taken as the background. The average of this was then subtracted from each sample. These values were then plotted directly or the ratio of Cy5 to BDP-FL or AF488 in each well was calculated. To calculate the percentage activation, the average MFI of the SNAP-treated cells in each experiment was obtained. The MFI of each untreated sample was then divided by this and multiplied by 100 to obtain the percent activation.
Following gating (Supplementary Figs.
3
Flow Cytometric Analysis of Cell Surface Markers
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The cells were stained with fluorescence-conjugated antibodies against MHC class II, CD40, CD80, and CD86 (all from eBioscience, San Diego, CA) for 30 min at 4 °C. The stained cells were assayed for fluorescence using the S1000EXi Flow Cytometer (Stratedigm, San Jose, CA). The data were analyzed using the FlowJo program (BD, Franklin Lakes, NJ).
4
Immunophenotyping of Murine Blood Cells
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The peripheral blood was collected, treated with RBC lysis buffer for 5 min, and centrifuged at 2000 rpm for 10 min. The resultant blood cells were stained with Abs against CD11b, Ly6G or Ly6C (eBioscience, San Diego, CA) at 4 °C for 30 min. Keratocytes were stained with anti-TLR2 Ab, anti-TLR3 Ab and anti-TLR4 Ab (Invitrogen, Waltham, MA). The stained cells were assessed for fluorescence using a S1000EXi Flow Cytometer (Stratedigm, San Jose, CA). The data were analyzed using the FlowJo program (Tree Star, Ashland, OR).
5
Single-Cell Isolation and Analysis of Lymph Node
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To obtain single-cell suspension, the bilateral cervical draining lymph nodes (DLNs) were minced between the frosted ends of two glass slides in the media containing RPMI-1640 (WelGENE, Daegu, Korea) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and were then filtered through a cell strainer. The resultant single-cell suspensions were stained with fluorescence-conjugated antibodies against CD4-PE cy7 eBioscience, Waltham, MA) and IFN-γ-FITC (eBioscience). For intracellular IFN-γ staining, the cells were prestimulated for 5 h at 37 °C with 5 μg/mL anti-CD3 (BD Pharmingen, San Diego, CA) and 5 μg/mL anti-CD28 (BD Pharmingen) in the presence of Cell Stimulation Cocktail including phorbol 12-myristate 13-acetate (PMA) and ionomycin (Cat No. 00-4970, eBioscience). The stained cells were assayed by a flow cytometer (S1000EXi Flow Cytometer, Stratedigm, San Jose, CA, USA) and were analyzed using FlowJo.
6
Antibody-Dependent Cellular Phagocytosis Assay
7
Multiparametric Flow Cytometry for T-cell Profiling
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Single cell suspensions were isolated from tissues and stained with fluorescence-conjugated antibodies against CD4-PE cy7 (Cat# 25-0041, eBioscience, Waltham, MA), IFN‐γ-FITC (Cat# 11-7311, eBioscience), and IL-17-APC (Cat# 17-7177, eBioscience). The stained cells were assayed by a flow cytometer (S1000EXi Flow Cytometer, Stratedigm, San Jose, CA) and analyzed using FlowJo program (Tree Star, Inc., Ashland, OR).
8
Prednisolone Effects on hCEC Apoptosis and Mitochondrial ROS
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The hCECs (1 × 105 cell per well) were seeded and cultured in SHEM in a 6-well plate for 24 h. The cells were then treated with prednisolone or solvent and further cultured for 3 days. For apoptosis assay, the cells were stained with a combination of PI and Annexin V (Cat no. 556547, FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen™, San Diego, CA) at room temperature for 5 min and analyzed by flow cytometry (S1000EXi Flow Cytometer, Stratedigm, Inc., San Jose, CA). For mitochondrial ROS measurements, the cells were stained with both CellROX dye (5 μM, CellROXTM Deep Red Reagent, Invitrogen) and MitoTracker Green dye (100 nM, MitoTracker Green FM Dye, Invitrogen) at 37 °C for 30 min, and analyzed for fluorescence using flow cytometry. The data obtained from flow cytometer were analyzed using Flowjo software (Tree Star, Ashland, OR).
9
Ploidy analysis of plant seedlings
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Ploidy analysis was performed as described in literature [34 (link)] protocol with slight modification; 20 mg of seedling at 30 DAS control and heat-treated were taken in test tubes containing 1 mL of ice cold Galbraith’s buffer (45 mM MgCl2, 20 mM MOPS (3-(N-morpholino)propanesulfonic acid), 30 mM sodium citrate, and 0.1% TritonX-100 at pH 7) and lysed by tissue lyser (1.5 min at 25 Hz). Homogenate was filtered through 40 µm nylon mesh. The filtrate was centrifuged at 200 g for 5 min to sediment the nuclei, and the pellet was suspended in 200 µL of Galbraith’s buffer. The samples were stained with 50 µg/mL propidium iodide containing 50 µg/mL RNase. After staining, samples were analyzed using Stratedigm S1000Exi flow cytometer (San Jose, CA, USA). Data analysis was done by FlowJo software (Version10) (Tree Star, Inc. Ashland, OR, USA).
10
Phenotyping Immune Cell Subsets
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The cells were stained with fluorescence-conjugated antibodies against HLA-DR, CD80, CD11b, and CD206 (eBioscience, San Diego, CA, USA), and fluorescence was measured using an S1000EXi Flow Cytometer (Stratedigm, San Jose, CA, USA). Data were analyzed using the FlowJo program (Tree Star, Inc., Ashland, OR, USA).
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