Cells were stained with various fluorophore-labeled antibodies from BD Biosciences (San Diego, CA), Biolegend (San Diego, CA), or Tonbo Biosciences (San Diego, CA) at 4°C. For intracellular staining, cells were fixed and permeabilized using the eBioscience FoxP3 Fixation/permeabilization buffer kit (for Nur77 and Ki67) or the BD Biosciences Cytofix/Cytoperm kit (for cytokines) according to manufacturer protocols. Biotinylated peptide-MHC monomers were obtained from the NIH Tetramer Facility (Atlanta, GA). Tetramers were made by conjugating the biotin-labeled monomers with PE-labeled or APC-labeled streptavidin (Agilent) according to protocols from the NIH tetramer facility. A tetramer with the WR97 mutation in Ld was generated by the NIH Tetramer Facility and used for staining the IE1-Ld tetramer specific populations in 11/14 of the Ld 97R mice. No significant differences were observed when using the Ld 97W vs. 97R tetramer. Data were acquired on BD LSR Fortessa and analyzed using FlowJo software (Ashland, OR). Statistical analyses were conducted using GraphPad Prism (San Diego, CA).
Foxp3 fixation permeabilization buffer kit
Manufactured by BD
The FoxP3 Fixation/permeabilization buffer kit is a laboratory tool designed for the intracellular staining and detection of the transcription factor FoxP3. The kit provides the necessary reagents for the fixation and permeabilization of cells, which is a crucial step in the analysis of intracellular proteins.
Automatically generated - may contain errors
2 protocols using foxp3 fixation permeabilization buffer kit
1
Multiparameter Flow Cytometry Analysis
2
Flow Cytometry Protocol for Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions extracted from the various tissues were plated out into flow cytometry tubes (Sarstedt) at a concentration of 10 6 cells per ml. Cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) and 1 μg/ml ionomycin (Sigma Aldrich) for 4 hours. For intracellular staining, 2 μM monensin (Sigma Aldrich) was added for the final 2 hours. For intracellular staining, cells were fixed and permeabilised using the Foxp3 fixation/permeabilization buffer kit (BD Biosciences). FcR receptor blocking antibodies were added for 15 min at 4°C and surface staining antibodies added together with live/dead stain (Fixable Live/Dead stains Thermo Fisher) and incubated for 20 mins at room temperature in the dark. Cells were acquired within 24 hours of staining. Fluorochromes were purchased from either BD Biosciences, Biolegend or Thermo Fisher. Fluorochromes used were CD4 (RM4-5), CD8α (53-6.7), CD45.
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!