The inhibition of the JAK-STAT pathway by the non-cytotoxic concentrations of jakinibs chosen for the present experiments according to previously published data was evaluated in terms of STAT1 phosphorylation at Y701. For this purpose, monocytes detached from polystyrene plates (300.000 cells/500 μL RPMI 10 % iFBS), were exposed for 1 h to reported non-cytotoxic concentrations of ITA (25 μM), BARI (200 nM), the JAK2 inhibitor AG490 (25 μM), and the tyrosine kinase inhibitor AG126 (12.5 μM); 0.5 % DMSO was used as a vehicle control. Cells were then stimulated with 2 ng/mL IFN-γ for 10 min, fixed, permeabilized, and stained with anti-STAT1 (pY701)-Alexa Fluor 647, according to the BD™PhosphoFlow protocol [48 (link)], and acquired on the BD LSRFortessa™ cell flow cytometer.
Lsrfortessa cell flow cytometer
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The LSRFortessa™ cell flow cytometer is a laboratory instrument used for analyzing and sorting cells. It utilizes flow cytometry technology to detect and measure various characteristics of cells, such as size, granularity, and the presence of specific proteins or markers on the cell surface. The LSRFortessa™ is designed to provide researchers with a reliable and precise tool for cell analysis and sorting applications.
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7 protocols using lsrfortessa cell flow cytometer
1
JAK-STAT Pathway Inhibition Assessed
2
Mitochondrial Assessment by FACS
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Fluorescence‐activated cell sorting (FACS) analysis of mitochondria‐localized fluorescent dyes was used to assess relative mitochondrial content and mitochondrial membrane potential with MTG (ThermoFisher) and TMRM (Invitrogen), respectively (De Luca et al., 2015 ; Dingley et al., 2012 ). Propidium iodide (ThermoFisher) was used to detect dead cells. Stained cells were analyzed using a BD LSRFortessa™ cell flow cytometer (BD Bioscience).
3
Isolation and Characterization of PBMCs
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Peripheral blood samples were collected from 26 healthy volunteers (7 women and 19 men) aged between 20 and 30 years. Blood samples were drawn into Vacutainer® EDTA tubes (for in vitro cell cultures) and onto glass beads for defibrination (for monocyte enrichment by adherence to plastic and subsequent isolation of EVs). Both samples were diluted 1:1 with DPBS and centrifuged on a density gradient using Histopaque (1077 g/L) at 900×g for 30 min at room temperature (RT); then, peripheral blood mononuclear cells (PBMCs) were removed and washed with DPBS. Cell number and viability were determined by trypan blue dye exclusion using a Neubauer chamber (viability was always ≥98 %). In addition, PBMCs isolated from defibrinated blood samples were stained with anti-CD14-RD1 conjugated mAb and acquired on LSRFortessa™ cell flow cytometer (BD Biosciences) to calculate the CD14+ monocyte concentration.
PBMCs were then resuspended in RPMI 1640 medium without antibiotics and supplemented with heat-inactivated FBS (56 °C for 40 min) (iFBS).
PBMCs were then resuspended in RPMI 1640 medium without antibiotics and supplemented with heat-inactivated FBS (56 °C for 40 min) (iFBS).
4
PBMCs Proliferation Assay with L-EVs and M-EVs
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PBMCs were stained with 1 μL of CFSE (5 μM) in 10 mL of PBS and incubated for 20 min at 37 °C and 5 % CO2. Subsequently, they were centrifuged at 600×g for 10 min, and the supernatant was removed. Next, cells were incubated with RPMI supplemented with 10 % iFBS for 40 m at 37 °C and 5 % CO2 to eliminate the excess CFSE dye. CFSE-stained PBMCs (1 × 105 cells per well in 200 μl of RPMI 1640 supplemented with 5 % v/v iFBS) were then seeded in 96-well cell culture plates. Cell proliferation was induced with 4 % v/v PHA (0.1 mg/mL) in the presence of 1.25 × 106 L-EVs and M-EVs released under treatment with jakinibs. After 6 h of PHA stimulation, supernatants were collected to quantify cytokines, and incubation went on to complete 96 h at 37 °C and 5 % CO2. Cells were finally stained with Life/Death-Vioblue to exclude dead cells and were labeled with fluorescent anti-CD69-PE, anti-CD25-Pe-Cy5, anti-CD8-PeCy5, and anti-CD3-PeCy7 mAbs. Cells were acquired on a BD LSRFortessa™ cell flow cytometer.
5
Monocyte Enrichment Protocol for Flow Cytometry
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PBMC suspensions (containing 3 × 105 CD14+ cells) were seeded per well in 48-well polystyrene tissue culture plates and resuspended in 500 μL of RPMI 1640 supplemented with 0.5 % v/v iFBS. Cells were incubated at 37 °C in a 5 % CO2 atmosphere for 2 h, and then washed with DPBS containing 0.5 % iFBS to remove non-adherent cells. Cells adhered to the plate were incubated in 500 μL of RPMI supplemented with 10 % iFBS at 37 °C and 5 % CO2. After 24 h, cells were detached with a glass scrapper and suspended in RPMI supplemented with 10 % iFBS. To verify monocyte enrichment and determine their purity, cells were stained with anti-CD19-V450, anti-CD3-PE-Cy7, and anti-CD56-PE mAbs and acquired on a BD LSRFortessa™ cell flow cytometer [47 (link)].
6
Jakinibs Modulate IFN-γ-Induced HLA-DR Expression
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The effect of jakinibs on IFN-γ-induced HLA-DR expression in peripheral blood monocytes was also evaluated. For this purpose, 3 × 105 PBMCs/well were seeded in 48-well cell culture plates and resuspended in 500 μl RPMI 1640 supplemented with 10 % iSBF, exposed to jakinibs and controls (at concentrations described above), for 1 h and stimulated with 2 ng/mL IFN-γ. After 24 h, cells were stained with Propidium iodide (PI) (to exclude dead cells), anti-HLA-DR-APCCy7, anti-CD19-V450, and anti-CD14-RD1 mAbs and acquired on the BD LSRFortessa™ cell flow cytometer.
7
Neutrophil Isolation and Activation Assay
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Neutrophils were isolated through density gradients following the protocol of Kuhns et al. [51 (link)]. Briefly, neutrophils (2 × 105 cells/300 μL) were deposited into 12 mm × 75 mm polyethylene tubes containing RPMI 1640 and stained with DHR123, at a final concentration of 5 nM, and incubated at 37 °C in a water bath for 10 min. Subsequently, cells were exposed to 1.75 × 106 L-EVs and M-EVs for 40 m at 37 °C. After incubation, neutrophils were stimulated with PMA, at a final concentration of 10 ng/mL, for 10 min. Cells were acquired on a BD LSRFortessa™ cell flow cytometer [52 (link)].
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