To assess BMDC activation status and viability after CS treatment, BMDCs were treated for 24 h. After treatment, cells were harvested and stained for CD11b BV421 (BioLegend, clone: M1/70), CD11c APC Fire 750 (BioLegend, clone: N418), I‐A/I‐E major histocompatibility complex (MHC) Class II V500 (BD Biosciences, clone: M5/114.15.2), CD40 PE‐Cy7 (BioLegend, clone: 3/23), CD80 PE (BD Pharmingen, clone: 16‐10‐A1), CD86 APC (eBiosciences, clone: GL1), Annexin V PE (BD Biosciences), and 7‐aminoactinomycin D (7AAD; BD Biosciences). Samples were analyzed on a Cytek DxP10 (Cytek Biosciences, Inc.) flow at the University of Nebraska‐Lincoln Flow Cytometry Service Center. Data were analyzed using FlowJo software (Becton, Dickinson and Company).
Dxp10
Manufactured by Cytek
Sourced in United States
The DxP10 is a flow cytometry instrument manufactured by Cytek. It is designed to analyze and measure the physical and chemical characteristics of particles, cells, or other biological samples in a fluid suspension. The DxP10 utilizes advanced optical and electronic technologies to detect and quantify multiple parameters simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b bv421, by BioLegend (2 mentions)
Cd86 apc, by Thermo Fisher Scientific (2 mentions)
Cd40 pe cy7, by BioLegend (2 mentions)
Cd11c apc fire 750, by BioLegend (2 mentions)
Cd80 pe, by BD (2 mentions)
Annexin 5 pe, by BD (2 mentions)
7 aminoactinomycin d, by BD (1 mentions)
7 aad, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Cd133 2 pe, by Miltenyi Biotec (1 mentions)
Cd44 pecy5, by Thermo Fisher Scientific (1 mentions)
Cd15 apc, by BD (1 mentions)
Annexin 5 fitc pi apoptosis assay kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using dxp10
1
Assessing BMDC Activation and Viability
2
Assessing BMDC Activation and Viability After CS Treatment
3
Cell Surface and Intracellular Flow Cytometry
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For cell surface flow cytometry, cells were washed with phosphate-buffered saline (PBS) and then incubated with indicated antibodies for 20 minutes at room temperature. After washing, cells were analyzed by fluorescence-activated cell sorting (FACS). For intracellular flow cytometry, cells were fixed in 3.2% paraformaldehyde for 10 minutes at 37°C, and permeabilized with 100% methanol for 30 minutes at −80°C. After washing, cells were incubated with indicated antibodies and then analyzed by FACS. Data were collected on a FACS Calibur (BD Biosciences) or a DxP10 (Cytek) flow cytometer and analyzed using FlowJo Software (v.10). All antibodies are listed in the Online Supplementary Methods.
4
Flow Cytometry Cell Viability Assay
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Detailed description can be found elsewhere [30 (link)]. Population compositions were measured by flow cytometry using a DxP10 (Cytek, Fremont, CA, USA). Fluorescent beads of known concentration (as determined by hemocytometer) were added to determine cell densities. A final 1:20,000 dilution of ToPro3 (Molecular Probes T-3605; Eugene, OR, USA) was used for each sample to determine live- and dead-cell densities. Analysis using FlowJo software showed obvious clustering of live and dead cells in the ToPro3 RedFL1 channel, with dead cells having a RedFL1 signal of >103. Dead-cell densities typically were never higher than 10% in all conditions tested.
5
Single-Cell Flow Cytometry Profiling
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The harvested cells were dissociated
into single cells with Accutase and then fixed with 4% PFA at room
temperature for 20 min. Single cells were stained with primary antibodies
(Table S1 ) at 4 °C overnight. After
washing the cells three times with 1% BSA in PBS, secondary antibodies
were added and incubated at room temperature for 2 h. Cells were washed
with 1% BSA in PBS and analyzed using a Cytek DxP10 flow cytometer.
Single-color and isotype controls served as compensation and negative
gating, respectively.
into single cells with Accutase and then fixed with 4% PFA at room
temperature for 20 min. Single cells were stained with primary antibodies
(
washing the cells three times with 1% BSA in PBS, secondary antibodies
were added and incubated at room temperature for 2 h. Cells were washed
with 1% BSA in PBS and analyzed using a Cytek DxP10 flow cytometer.
Single-color and isotype controls served as compensation and negative
gating, respectively.
6
Multiparameter Flow Cytometry Analysis
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We used the following antibodies: CD44-PE-Cy5 (1:100) (eBioscience), CD15-APC (1:5) (BD), CD133/2-PE (1:50) (Miltenyi Biotec). Annexin V-FITC Apoptosis Detection Kit was obtained from eBioscience. Staining was quantified by flow cytometry (Cytek DxP10).
7
Annexin V-FITC/PI Apoptosis Assay
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The Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) was employed to evaluate the apoptosis of the HL-60 and THP-1 cells refer-ring to the directions of the manufacturer. The apoptotic cells were assessed using a flow cytometer (model: DxP 10; Cytek Biosciences).
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