Up to 2 µg of lEVs were blocked for 15 min at RT in 20 µl PBS + 1% EV-depleted FCS and stained with fluorescently-labeled antibodies directed against EpCAM (#324208, 30 ng/sample), ROR1 (#357803, 2.5 ng/sample, both from Biolegend), ROR2 (#FAB20641G, 12.5 ng/sample, R&D systems), or corresponding isotype controls at the same concentration (#400321 and #400113 from Biolegend, #IC003G from R&D systems) for 20 min at RT. Fluorescence was recorded on the FACSymphony A1 (BD) flow cytometer and the percentage of positive events in the lEV gate was determined in relation to the respective isotype control. The submicron bead calibration kit (#832, Bangs Laboratories) was used to define the gate for lEVs. For EV uptake studies, cells were pretreated with Dynasore (12.5 µM) for 2 h prior to the addition of DiR-labeled EVs (10 µg/ml) for 24 h. Cells were washed twice with PBS and the mean fluorescence DiR intensity of single cells was recorded on the FACSymphony A1 (BD) flow cytometer. Data was analyzed with FACS Diva (version 9.0.2, BD) and FlowJo (version 10.6.1, BD) software. Flow cytometer acquisition settings were maintained for all samples, including triggering threshold, voltages, and flow rate.
Facsymphony a1
Manufactured by BD
Sourced in United States
The FACSymphony A1 is a flow cytometry system designed for high-performance cell analysis and sorting. It features advanced optics, fluidics, and electronics to deliver accurate and reliable results. The core function of the FACSymphony A1 is to enable the identification, enumeration, and isolation of specific cell populations from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Submicron bead calibration kit, by Bangs Laboratories (1 mentions)
Ic003g, by R&D Systems (1 mentions)
Fab20641g, by R&D Systems (1 mentions)
Facsdiva, by BD (1 mentions)
Sandwich elisa, by Shino-Test (1 mentions)
Cobas e601 module, by Roche (1 mentions)
Collagenase, by Merck Group (1 mentions)
Lypd8, by BioLegend (1 mentions)
Hepes buffer, by Lonza (1 mentions)
Live dead fixable yellow, by Thermo Fisher Scientific (1 mentions)
Epcam, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Anti cd45 microbeads, by Miltenyi Biotec (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Fbs aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Clone cd28, by BioLegend (1 mentions)
7 protocols using facsymphony a1
1
Characterization of Extracellular Vesicles
2
HMGB1 and Immune Marker Profiling in Breast Cancer
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Fasting peripheral blood was collected in tubes containing ethylenediaminetetraacetic acid before and after NACT. The serum was separated by centrifugation at 3000 rpm for 10 min at room temperature, aliquoted, and stored at − 80 °C. The HMGB1 concentration was measured using the Sandwich ELISA of Shino-Test (Tokyo, Japan). The samples were added to the wells of microtiter dishes coated with anti-HMGB1 antibody. After incubation for 24 h at 4 °C, the plates were washed and incubated with a second enzyme-labelled antibody for 1 h at room temperature. A colored solution was added for 30 min, and the HMGB1 concentration was spectrophotometrically determined at 450 nm using a standard curve prepared with the kit. CEA, CK19, CA15-3, and E-cadherin levels were measured by enzymatic chemiluminescent immunoassay on an automatic immunoassay analyzer (Cobas e601 module, Roche, Sweden) using enzyme-linked immunosorbent assay (ELISA) kits from CanAg Diagnostics, Sweden and Roche Diagnostics, Sweden, according to the manufacturer’s instructions. Th17, CD4+CD25+ Tregs, and Treg cells were assessed using a flow cytometer (FACSymphony A1, BD Biosciences, USA). All samples were assayed in triplicates.
3
Isolation and Analysis of mTECs
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mTECs were isolated, analyzed, and sorted as previously described (Michelson et al., 2022a (link)). Thymi were finely chopped, the lymphocyte-rich supernatant was removed, and the thymic pieces were incubated at 37°C in DMEM supplemented with 2% FCS, 25 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Lonza), 0.5 mg/ml collagenase (Sigma-Aldrich), and 0.1 mg/ml DNaseI (Sigma-Aldrich) for 15 min, then in the same medium with 0.5 mg/ml collagenase/dispase (Roche) for 15 min. To dissociate cell–cell interactions, 10 mM EDTA was added. To prepurify mTECs, cells were incubated with anti-CD45 microbeads (Miltenyi) for 15 min and then CD45+ cells were depleted using MACS LS columns (Miltenyi). Cells were then stained with antibodies against EpCAM, CD45, Ly51, A/E, Lypd8 (all Biolegend), and/or GP2 (MBL). DAPI (Sigma-Aldrich) and Fixable Yellow Live/Dead (Invitrogen) were used for dead cell exclusion. mTECs were defined as live CD45− EpCAM+ Ly51− cells and further gated as mTEChi or mTEClo based on MHCII levels. Flow cytometry was performed on LSRII or FACSymphony A1 instruments (BD), and cell sorting was performed on a FACSAria cell sorter (BD). Flow cytometry data were analyzed with Flowjo (BD).
4
CD44 Expression in Gastric Cancer Cells
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MKN-45 miR−18-oe, MKN-45 miR−18-kd, AGS miR−18-oe, AGS miR−18-kd and vector cells were harvested and washed three times with PBS and stained with anti-CD44 (750,211, BD Biosciences). The flow cytometer (FACSymphony™ A1, BD Biosciences) was used for determination of CD44+ sub-population. The results of the experiment were analyzed through flow cytometry software as previously described [34 (link)].
5
T Cell Proliferation Assay with OX40 Activation
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T cells (1 × 106 cells/mL in DPBS) were labeled with 5 μM of CellTrace Violet (Life Technologies) and incubated at 37 °C for 20 min. Medium containing 2% FBS was added (five times the original volume of the staining medium) and centrifuged once to remove free dye. The cell pellet was resuspended in 10% FBS-AIM-V medium (Life Technologies) at 1 × 106 cells/mL, and 100 μL aliquots of the cells were added into wells precoated with 100 μL of anti-CD3 antibody (0.5 μg/mL, clone UCHT-1, Biolegend) and fusion antibodies to OX40 (0.25, 0.5, 1.0 and 2.0 μg/mL); soluble anti-CD28 (1 μg/mL; clone CD28.2, Biolegend) was added to the wells containing T cells. T cells in culture medium alone served as the non-stimulated cell control. The cells were then kept at 37 °C in a CO2 incubator for 5 days. They were then harvested and the reduction of CellTrace Violet (indicating cell proliferation) was assessed via flow cytometry (BD FACSymphony A1, BD biosciences).
6
Multiparameter Flow Cytometry Staining
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Cells were stained for 20 min on ice in MACS buffer (2% FCS in PBS with 1 mM EDTA) at 0.5 to 1 × 106 cells per well in 96-well round-bottom plates unless otherwise specified. The following monoclonal antibodies were used: B220–BV785 (BioLegend), ICAM1–biotin (BD), followed by streptavidin–BV711 (Fisher), CD45.2–PerCP-Cy5.5 (Tonbo), IgMa–FITC (Fisher), CD40–PE (Fisher), CD23–PE-Cy7 (BioLegend), CD45.1–BV605 (BioLegend), CD40L–PE (BioLegend), and CD40L–biotin (eBioscience), followed by streptavidin–A647 (Fisher). Dead cells were excluded using Fixable Viability Dye eFluor780 (eBioscience no. 65-0865-18). All samples were run on a BD LSRII or BD FACSymphony A1 at 5,000 to 10,000 events per second. Flow cytometry data were analyzed using FlowJo (v10.8.1).
7
Fusion Antibodies' Impact on T Cell Survival
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To determine the effect of fusion antibodies on T cell survival, T cells (1 × 106 cells/mL of 10% FBS-AIM-V medium) that had been activated with immobilized anti-CD3 antibody and fusion antibodies in the presence of soluble anti-CD28 were cultured at 37 °C in CO2 incubator for 5 days. The cells were harvested, washed twice with DPBS, then stained with 0.5 µM ethidium homodimer D-1 (EthD-1) (Invitrogen) and 400 nM Apotracker green (Biolegend). Percentages of living cells, total dead cells, necrotic cells, and early/late apoptotic cells were assessed via flow cytometry (BD FACSymphony A1, BD biosciences) and compared with the CD3-CD28-activated T cells in the medium alone (no immobilized fusion antibodies)
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