To stain for transcription factors, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, California, USA), according to the manufacturer's instructions. For intracellular flow cytometric (ICF) staining of cytokines cells were re-stimulated for 4 h with phorbol 12,13-dibutyrate (PDBu)/ionomycin (50 ng/ml and 500 ng/ml, respectively) in the presence of Golgi Stop (BD Bioscience San Jose, California USA). Thereafter, the cells were washed, stained for surface antigens and subsequently fixed and permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences). Data were acquired on a FACSCalibur instrument (BD Biosciences) and analyzed with FlowLogic software (eBioscience). The following antibodies were used: PE-Cy7- or FITC-conjugated anti-CD4 (RM.4–5), PE-conjugated anti-IL-17A (TC11-18H10), and APC- or PE-Cy7-conjugated anti-IFN-γ (XMG1.2) from BD Pharmingen; PE-conjugated anti-T-bet (4-B10) and APC-conjugated anti-RORγt (AFKJS-9) from eBioscience.
Flowlogic software
Manufactured by Thermo Fisher Scientific
FlowLogic is a software package designed for the analysis and visualization of flow cytometry data. It provides tools for data management, gating, and statistical analysis of multi-parameter flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Anti cd3 pe cy7, by BioLegend (2 mentions)
Facsverse, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti cd24 fitc, by BioLegend (1 mentions)
Anti cd5 percp cy5, by BioLegend (1 mentions)
Tcrβ pe cy7, by BioLegend (1 mentions)
Anti cd69 pe, by BioLegend (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti cd44 pe cy7, by BioLegend (1 mentions)
Anti cd25 apc, by BioLegend (1 mentions)
Anti b220 pe, by BioLegend (1 mentions)
Anti cd8 apc, by BioLegend (1 mentions)
Foxp3 pe staining buffer set, by Thermo Fisher Scientific (1 mentions)
Bio plex pro mouse cytokine il 2 set, by Bio-Rad (1 mentions)
Bio plex suspension array system, by Bio-Rad (1 mentions)
Anti cd16 32, by BD (1 mentions)
Hla dr, by BD (1 mentions)
6 protocols using flowlogic software
1
Intracellular Cytokine Staining Protocol
2
Multiparameter Flow Cytometry Analysis
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Single cell suspensions from the spleen, lymph node and thymus were prepared and stained after a washing step for surface marker expression with the following fluorochrome conjugated antibodies: anti-CD3-PECy7, anti-CD4-FITC, anti-CD8-APC and anti-B220-PE, (all from Biolegend). For the staining of activation markers, cells were pre-activated for 24 h with stimulating antibodies (aCD3 and aCD28) and then stained with the following antibodies: anti-CD25-APC, anti-CD44-PECy7 and anti-CD69-PE (all from Biolegend). For analyses of thymocytes the following antibodies were used: anti-CD24-FITC, anti-CD5-PerCP Cy5.5 and TCRβ-Pe Cy7 (all from Biolegend).
For the staining of intracellular FoxP3, the cells were fixed and subsequently permeabilized to the staining of surface antigens. The FoxP3 FITC staining buffer set (eBioscience) was used for the detection of Foxp3. Data were acquired on a FACSCalibur (CellQuest, BD Biosciences) and analyzed with FlowLogic software (eBioscience).
For the staining of intracellular FoxP3, the cells were fixed and subsequently permeabilized to the staining of surface antigens. The FoxP3 FITC staining buffer set (eBioscience) was used for the detection of Foxp3. Data were acquired on a FACSCalibur (CellQuest, BD Biosciences) and analyzed with FlowLogic software (eBioscience).
3
Multi-Parametric Flow Cytometry Analysis
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Surface staining (including pre-incubation with FcR block; anti-CD16/32; BD Biosciences) was performed as described previously [41 ]. The following antibodies were used: anti-CD25 PE or APC, anti-CD4 FITC or APC, and anti-CD3 PECy7 (all from Biolegend). For the staining of intracellular antigens, the cells were fixed and subsequently permeabilized to the staining of surface antigens. The FoxP3 PE staining buffer set (eBiosciences) was used for the detection of Foxp3. For PKCθ fixation buffer and intracellular staining permeabilization wash buffer obtained from Biolegend and a mouse monoclonal anti-human PKCθ antibody (BD Biosciences) were used. Data were acquired on a FACSCalibur (CellQuest, BD Biosciences) and analyzed with FlowLogic software (eBioscience). The concentration of secreted IL-2 in the culture supernatants was measured by Luminex xMAP Technology using a Bio-Plex Pro Mouse Cytokine IL-2 Set (No. 171G5003M), according to the manufacturer´s instructions, on a Bio-Plex suspension array system (all Bio-Rad).
4
Multicolor FACS Analysis of HIV-Exposed DCs
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Multicolor FACS analyses of HIV-exposed DCs using anti-human CD83, CD86, CCR7, CD40, HLA-ABC, HLA-DR (all BD Biosciences) were performed as described [32 (link)] on a FACS Verse flow cytometer (BD Biosciences). Data were analyzed using FlowLogic Software (Affymetrix).
5
Pluripotency Marker and Embryoid Body Characterization
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For pluripotent marker and embryoid body experiments (Fig. 6) cells that were cultured for a minimum of 8 passages under RGA were compared to conventional FGA (daily fed) cultures.
iPSC were harvested with TrypLE™ (Life Technologies) followed by centrifugation and resuspension in PBS. All centrifugation steps for flow cytometry were carried out for 5 min at 250g at 4 °C. Cells were then prepared for flow cytometry using a Fixation Buffer and a Permeabilization Wash Buffer (Cat. 421,002 and 420801 BioLegend®) according to the manufacturer's instructions. Primary antibodies were incubated in Permeabilization Wash Buffer for 30 min followed by a washing step in Permeabilization Wash Buffer. Secondary antibody (for Oct3/4 labeled samples only) was incubated for 20 min in Permeabilization Wash Buffer followed by a washing step. Finally, cells were resuspended in PBS for flow cytometric analysis. The following antibodies and dilutions were used: Anti-Oct3/4 (Santa Cruz Biotechnologies,) 1:50, anti-Alexa Fluor® 647-Tra-1-60-R (BioLegend®) 1:10 and anti-Alexa Fluor® 488-Mouse (Molecular Probes,) 1:100. Multi-color FACS analyses were performed on a FACS Verse™ flow cytometer (BD Biosciences). Data were analyzed using FlowLogic® Software (Affymetrix).
iPSC were harvested with TrypLE™ (Life Technologies) followed by centrifugation and resuspension in PBS. All centrifugation steps for flow cytometry were carried out for 5 min at 250g at 4 °C. Cells were then prepared for flow cytometry using a Fixation Buffer and a Permeabilization Wash Buffer (Cat. 421,002 and 420801 BioLegend®) according to the manufacturer's instructions. Primary antibodies were incubated in Permeabilization Wash Buffer for 30 min followed by a washing step in Permeabilization Wash Buffer. Secondary antibody (for Oct3/4 labeled samples only) was incubated for 20 min in Permeabilization Wash Buffer followed by a washing step. Finally, cells were resuspended in PBS for flow cytometric analysis. The following antibodies and dilutions were used: Anti-Oct3/4 (Santa Cruz Biotechnologies,) 1:50, anti-Alexa Fluor® 647-Tra-1-60-R (BioLegend®) 1:10 and anti-Alexa Fluor® 488-Mouse (Molecular Probes,) 1:100. Multi-color FACS analyses were performed on a FACS Verse™ flow cytometer (BD Biosciences). Data were analyzed using FlowLogic® Software (Affymetrix).
6
Cell Cycle Analysis via Flow Cytometry
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Cells were washed in PBS and harvested via TrypLE™ incubation. Cells were fixed in 70% ethanol for 30 min or overnight at 4 °C. After fixation cells were washed 3 times in PBS and re-suspended in PI/ RNAse Staining buffer (BD Pharmingen™). Cell cycle analysis was carried out after 20 min of propidium iodide staining on a FACS Verse™ flow cytometer (BD Biosciences) and data were analyzed using FlowLogic® Software (Affymetrix).
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