BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (554714; BD Biosciences, San Jose, CA, USA) was used in flow cytometry to detect the expression of the various aforementioned smooth muscle cell markers by SHED-derived SMCs. Cell dissociation solution (C5914; Sigma-Aldrich) was used to disperse the SHED-derived SMCs. After fixation and permeabilization, cells were incubated for 2 hours with primary antibodies against α-SMA (ab124964; Abcam), SM22 alpha (ab14106; Abcam), smooth muscle Myosin heavy chain 11 (ab683; Abcam), Calponin (ab46794; Abcam), and then washed with PBS. This was followed by incubation with the appropriate secondary antibodies: goat anti-mouse IgG H&L (Alexa Fluor® 488) pre-adsorbed (ab150117; Abcam) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077; Abcam). In this study, mouse IgG1, kappa monoclonal (ab170190; Abcam) and rabbit IgG, monoclonal [EPR25A] (ab172730; Abcam) were utilized as the isotype control. Flow cytometry data was analyzed with FACSVerse software (BD Biosciences).
Facsverse software
Manufactured by BD
Sourced in United States
FACSVerse software is a data analysis and visualization tool designed for use with BD flow cytometry instruments. It provides capabilities for data acquisition, analysis, and presentation of flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Ab14106, by Abcam (2 mentions)
Ab124964, by Abcam (2 mentions)
Ab170190, by Abcam (2 mentions)
Ab150117, by Abcam (2 mentions)
Ab683, by Abcam (2 mentions)
Ab46794, by Abcam (2 mentions)
Ab172730, by Abcam (2 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (2 mentions)
C5914, by Merck Group (2 mentions)
Alexa fluor 488, by Abcam (2 mentions)
Verse cytometer, by BD (2 mentions)
Ab150077, by Abcam (1 mentions)
Facsverse cytometer, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Anti hla dr percp cy5.5 clone g46 6, by BD (1 mentions)
Prism version 6, by GraphPad (1 mentions)
Cyto x cytoperm, by BD (1 mentions)
Anti hla dr apc clone g46 6, by BD (1 mentions)
Percp cy5.5 anti cd4 clone okt4, by BioLegend (1 mentions)
5 protocols using facsverse software
1
Flow Cytometric Analysis of SHED-Derived Smooth Muscle Cell Markers
2
Quantifying Apoptosis via Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect apoptosis, Annexin V-FITC (fluorescein isothiocyanate) and PI staining were performed, and cells were evaluated using a FACSVerse cytometer (BD, San Jose, CA, USA) and an Annexin V-FITC Apoptosis Detection Kit (BD, San Jose, CA, USA) following the manufacturer’s protocol. In short, cells were treated with plasma and incubated for 24 h, after which they were collected, washed with Dulbecco’s phosphate-buffered saline (DPBS), stained with Annexin V-FITC and PI and analyzed using flow cytometry. FACSVerse software (BD Bioscience, San Jose, CA, USA) was used to analyze the percentage of cells in four populations, including FITC−/PI− (living cells), FITC+/PI− (early apoptotic cells), FITC+/PI− (late apoptotic cells), and FITC−/PI+ (necrotic cells).
3
Smooth Muscle Cell Marker Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed as previously described using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (554714; BD Biosciences) to detect the expression of the various aforementioned smooth muscle cell markers by SHED-derived smooth muscle cells (SMCs). A cell dissociation solution (C5914; Sigma-Aldrich) was used to disperse the SHED-derived SMCs. After fixation and permeabilization, cells were incubated for 2 h with primary antibodies against α-smooth muscle actin (α-SMA) (ab124964; Abcam, Cambridge, MA, USA), SM22 alpha (ab14106; Abcam), smooth muscle myosin heavy chain 11 (ab683; Abcam), and calponin (ab46794; Abcam), and then washed with phosphate-buffered saline (PBS). This was followed by incubation with the appropriate secondary antibodies: goat anti-mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117; Abcam) and goat anti-rabbit IgG H&L (Alexa Fluor® 488). In this study, mouse IgG1, kappa monoclonal (ab170190; Abcam), and rabbit IgG monoclonal [EPR25A] (ab172730; Abcam) were used as isotype controls. Flow cytometry data were analyzed using the FACSVerse software (BD Biosciences).
4
Multiparameter Flow Cytometric Analysis of T Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six-parameter flow cytometric analysis was performed on whole blood, IELs, and T cells from LNs according to standard procedures (24 (link)) using a panel of monoclonal antibodies (mAbs) that were originally designed to detect human molecules but that we and others have shown to be cross-reactive with rhesus monkey (3 (link), 19 (link), 25 (link), 26 (link)). The antibodies used were as follows: anti-CD3-FITC (clone SP34), anti-CD4-APC-H7 (clone L200), anti-CD8-PE-Cy7 (clone RPA-T8), anti-CD69-PE (clone FN50), and anti-HLA-DR-PerCP-Cy5.5 (clone G46-6) (all from BD Pharmingen). Isotype antibody was used for negative control of CD69 and HLA-DR expression. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA). The CD4+ and CD8+ T cell percentages were based on the CD3+ T cells, and the activation markers on CD4+ and CD8+ T cells were based on CD4+CD3+ and CD8+CD3+ T cells, respectively.
5
Multiparameter Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six-parameter ow cytometric analysis was performed on whole blood, BM-, LN-and spleen-derived cells according to standard procedures using a panel of mAbs purchased from BD Biosciences (Pharmingen, San Diego, CA) as follows: anti-CD3-APC-Cy7 (clone SP34), anti-CD8-PE (clone RPA-T8), anti-Ki-67-PE-Cy7 (clone B56), anti-CD14-PE (clone M5E2), anti-CD16-FITC (clone 3G8), and anti-HLA-DR-APC (clone G46-6). The anti-CD4-PerCP-Cy5.5 (clone OKT4) was purchased from Biolegend (San Diego, CA, USA). Isotype antibody was used for a negative control of CD4, Ki67, HLA-DR, CD14 and CD16 expression. Intracellular staining for Ki-67 was performed at room temperature for 30 minutes following permeabilization with cyto x/cytoperm (BD Bioscience). Flow cytometric acquisition and analysis were performed on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA) and graphs were prepared using Prism version 6.0 (GraphPad).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!