Cells were pre-treated with ASOs for three days before the addition of DPI, MET, or PER. Three days later, cells were harvested, washed, and stained using the Vybrant Apoptosis Kit (ThermoFisher, Waltham, MA) according to the manufacturer’s protocol. Stained samples were then examined on a Becton Dickenson FACSVerse flow cytometer and analyzed using FlowJo software.
Facsverse
Manufactured by FlowJo
The FACSVerse is a flow cytometry instrument designed for high-throughput sample analysis. It is capable of detecting and analyzing multiple parameters of individual cells or particles within a sample. The core function of the FACSVerse is to provide researchers with a reliable and efficient tool for collecting and processing flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by FlowJo (1 mentions)
Jagged1, by R&D Systems (1 mentions)
Cd163, by R&D Systems (1 mentions)
Cd4 v500, by BD (1 mentions)
Cd8 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd45 apc, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd11b antibody, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti gr1, by BD (1 mentions)
Pe conjugated anti cd11c, by Thermo Fisher Scientific (1 mentions)
P ask1, by Abcam (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
Trypsin, by Beyotime (1 mentions)
5 protocols using facsverse
1
Apoptosis Assay with Flow Cytometry
2
Characterization of TLR2/1 activated Macrophages
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Cells were harvested after 48 hours incubation at 37°Celsius in 7%CO2. Surface expression of protein was determined using specific antibodies: CD209 (Becton Dickinson), CD40 (Becton Dickinson), CD1a (Becton Dickinson), CD163 (R&D systems), Jagged1 (R&D systems), CD14 (Becton Dickinson) and IgG controls (Becton Dickinson). Phosphorylated STAT-1 levels were determined using Anti-Human phospho-STAT1 (eBiosciences). Cytometric Bead Arrays (CBA) were used to characterize TLR2/1R activated CD14+MΦ supernatants. CBAs were performed on 50μL of supernatant that was harvested after 24 hours of incubation. Supernatants were tested for the presence of MIP1-β, IL-6 and TNF-α. CBA Flex kits were obtained from Becton Dickinson and performed according to manufacturer’s recommendations. Samples were acquired using FacsCalibur and FacsVerse flow cytometers and FCS files were analyzed using FlowJo software.
3
Immunophenotyping of Brain and Spinal Cord Cells
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Homogenized brain and spinal cord cells were incubated with FC-block (BD 553141) for 15 min at 4°, then stained for 20 min at 4°C with specific antibodies (CD8 PerCPCy5.5 [eBiosciences 45-0081-80], CD45 APC [eBiosciences 17-0451-81], CD4 V500 [BD 560782], CD3 PB [Biolegend 100214]) diluted 1:200 in PBS/2% FCS. Data acquisition was performed on a FACSVerse and data analysis was conducted using the FlowJo 10.0.8r1 software.
4
Neutrophil Evaluation After TB Challenge
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The neutrophils from the vaccinated animals were evaluated 30 days after the challenge with M. tuberculosis. The splenocytes were obtained as described above. The cells were incubated for 30 min with APC-conjugated anti-GR1 (BD PharMingen), PercP-conjugated anti-CD14, PE-conjugated anti-CD11c and FITC-conjugated anti-CD11b antibodies (eBioscience). Then, the cells were washed with PBS and fixed with paraformaldehyde using Perm Fix (BD Cytofix). A total of 100,000 events were acquired using BD Biosciences FACSVerseTM and analyzed with the FlowJo 9.0 Software. The neutrophils percentage was determined evaluating singlets that were GR-1+, CD11c-, and CD14-. The total number of neutrophils was obtained multiplying the percentage of neutrophils by the total number of splenocytes from each animal.
5
Phosphorylated ASK1 Quantification
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For collection, TC-1 cells were washed with PBS, then trypsin (Beyotime, China) was used. The cells were incubated with Fix/Permed by in a Transcription Factor Buffer Set (BD, 562574) at room temperature in the dark for 15 min.
Subsequently, the cells were incubated with p-ASK1 (Abcam, ab278785) for 30 min at room temperature in the dark for 30 min. BD Biosciences FACSVerseTM was used to test all the samples, and FlowJo 10.4 software was used to analyze the results.
Subsequently, the cells were incubated with p-ASK1 (Abcam, ab278785) for 30 min at room temperature in the dark for 30 min. BD Biosciences FACSVerseTM was used to test all the samples, and FlowJo 10.4 software was used to analyze the results.
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