To characterize the isolated basal cells, flow cytometry analysis was performed. In brief, cells at passage 3 were collected, blocked withFBS and permeabilized by permeabilizing buffer (eBioscience) for 20 min. Thereafter, cells were incubated with anti-human CD10 antibody (FITC-conjugated; F0826; Dako) for 30 min. After three wash with phosphate-buffered saline (PBS), the percent of CD10 positive BCC was calculated by using BD FACSCalibur apparatus and FlowJo software (version 7.6.1.).
Permeabilizing buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Permeabilizing buffer is a laboratory reagent used to temporarily increase the permeability of cell membranes. It facilitates the introduction of substances, such as dyes, antibodies, or other molecules, into the interior of cells for analysis or experimentation purposes. The buffer composition is designed to maintain cell integrity while enabling controlled permeabilization.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Perm buffer 3, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Version 7, by FlowJo (1 mentions)
Cytofix fixation buffer, by BD (1 mentions)
Fixation buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by BD (1 mentions)
Anti mouse cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Anti cd80 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd11c pe, by Thermo Fisher Scientific (1 mentions)
Anti cd11c apc, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Fitc conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti tnf α antibody, by Abcam (1 mentions)
6 protocols using permeabilizing buffer
1
Flow Cytometry Analysis of Isolated Basal Cells
2
Quantifying RBD-specific T Cell Immunity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Intracellular cytokine staining (ICS), freshly isolated mouse splenocytes or LN cells were stimulated with an overlapping peptide pool of RBDWT (10 μg/mL) in the presence of Brefeldin A (Invitrogen, USA) for 5 h at 37°C, 5% CO2. The cells were harvested and stained with anti-CD3, anti-CD4 and anti-CD8α surface markers, and subsequently fixed and permeabilized in permeabilizing buffer (eBiosciences, USA) and stained with fluorescence-conjugated anti-IFN-γ, anti-TNF-α, anti-IL-2 and anti-IL-4 antibodies.
For virus-specific CTL frequency analysis, PE-labeled H-2Db tetramer containing the “S” epitope (SVLYNSASF) of RBD (HELIXGEN, Guang Zhou, China) was used. Freshly prepared mouse splenocytes or LN cells were stained with anti-CD45-AF700, CD3-FITC, CD8-APC,PE-tetramer, followed by FACS analysis. All fluorescence-labeled Abs were from BioLegend, and the stained lymphocytes were analysed on Attune NxT Flow Cytometer (ThermoFisher, USA).
For virus-specific CTL frequency analysis, PE-labeled H-2Db tetramer containing the “S” epitope (SVLYNSASF) of RBD (HELIXGEN, Guang Zhou, China) was used. Freshly prepared mouse splenocytes or LN cells were stained with anti-CD45-AF700, CD3-FITC, CD8-APC,PE-tetramer, followed by FACS analysis. All fluorescence-labeled Abs were from BioLegend, and the stained lymphocytes were analysed on Attune NxT Flow Cytometer (ThermoFisher, USA).
3
Intracellular Phospho-AKT and FOXO3 Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 3 days of transwell co‐culture, T cells were isolated and restimulated with plate‐bound anti‐CD3 (2 ug/ml; BD) for 10 minutes in a water bath at 37°C. The stimulation was attenuated by fixation with BD cytofix‐fixation buffer (BD) for 10 minutes at 37°C. Cells were then permeabilized with Perm Buffer III (BD) for 30 minutes on ice before staining with anti‐p‐AKT (pT308) antibody (#562465; BD) for 1 hour. For intracellular FOXO3 staining, cells were fixed using fixation buffer (eBioscience) for 30 minutes. Cells were washed twice before permeabilizing with permeabilizing buffer (eBioscience) for 30 minutes at room temperature. Permeabilized cells were treated with anti‐FOXO3 (#2497; Cell Signaling Technology Europe BV, Leiden, The Netherlands) for 20 minutes, followed by 20 ug/ml goat anti‐rabbit Alexa Fluor 647 secondary antibody (#4414; Cell Signaling Technology) for 20 minutes. Cells were washed twice with FACS buffer before acquiring on a BD LSRFortessa (BD).
Fifty thousand gated events were recorded per sample and analyzed using FlowJo Version 7.6 (FlowJo, Ashland, OH).
Fifty thousand gated events were recorded per sample and analyzed using FlowJo Version 7.6 (FlowJo, Ashland, OH).
4
HSV-1 Intracellular ICP27 Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DCs were harvested at the time of assay and washed once with FACS buffer (D-PBS containing 0.5% BSA and 2 mM EDTA), followed by blocking non-specific binding with anti-mouse CD16/CD32 (eBioscience, San Diego, CA). To examine cell surface molecules, cells were stained with isotype-matched antibodies, anti-CD11c-PE or anti-CD11c-APC, anti-CD40-FITC, anti-CD80-FITC and anti-CD86-FITC antibodies (eBioscience, San Diego, CA) for 30 min on ice. For intracellular ICP27 staining, cells were fixed in 4% paraformaldehyde (Sigma) and permeabilized in permeabilizing buffer (eBioscience, San Diego, CA). Cells were blocked with 5% normal goat serum (Sigma), incubated with anti-HSV-1 ICP27 (Virusys, Sykesville, MD), and stained with goat anti-mouse FITC-conjugated antibody (Santa Cruz Biotech, CA). Samples were processed and screened using a FACSCalibur fluorescence-activated cell sorter (FACS) and data were analyzed using FlowJo VX software.
5
Immunofluorescence Staining for Intracellular TNF-α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We also performed immunofluorescence staining (IF) to detect the intracellular level of TNF-α after exposure to the DHA, Pal, and insulin. In brief, 1 × 104 cells were placed in each well of 8 well slide chambers (SPL). After reaching 70–80% confluence, cells were subjected to the experimental protocol at the respective time point. Cells were fixed with 4% PFA and permeabilized using permeabilizing buffer (Ref no: 00–0055-56; eBioscience; USA) for 20 min. Cells were then incubated with anti-TNF-α antibody (dilution: 1:100; Cat no: ab34674; Abcam) for 1 h followed by PBS wash (3 × 10 min). After that, we added FITC-conjugated secondary antibody (1: 1000; eBioscience; USA) for 1 h at RT. After PBS wash, the nuclei were stained with 1 μg/ml for 30 s [18 (link)].
6
Apoptosis Quantification in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the occurrence of apoptosis in cells from different groups, the percent of HUVECs entering apoptosis was determined by using flow cytometry analysis. For this purpose, 2× 105 HUVECs were seeded in each well of 24-well plates and allowed to reach 70–80% confluence. The cells were subjected to the treatment protocol. After 48 h, cells were detached using 0.25% Trypsin-EDTA solution and washed twice with PBS. After that, cells were permeabilized using a permeabilizing buffer (eBioscience, USA) at room temperature for 30 min. In the current experiment, we incubated cells from different groups with 2.5 μl FITC-tagged Annexin-V antibody at 4̊C for 30 min. After twice washing with PBS, we added 2.5 μl propidium iodide and incubated cells for b5 minutes. The cells were analyzed by using the BD FACSCalibur® system and FlowJo software ver.7.6.1 [20 ].
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!