Mice were perfused for the analysis of brain sequestered cells. Brains were digested in RPMI containing collagenase (1.6 mg/mL; type IV; Sigma Aldrich) and DNaseI (200 μg/mL; Sigma Aldrich) at 37 °C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare). Debris was filtered out using a 70 μm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability maker according to the manufacturer’s protocol (Invitrogen). The cells were blocked and labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), APC anti-CD4 (eBioscience; RM4-5) and PerCP-Cy5.5 anti-CD8 (eBioscience; 53–6.7). Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using the FACS Diva 6.0 software.
Percp cy5.5 anti cd8
Manufactured by Thermo Fisher Scientific
PerCP-Cy5.5 anti-CD8 is a fluorescently labeled antibody that binds to the CD8 antigen. It is used for the detection and analysis of CD8+ cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (2 mentions)
Anti cd45 fitc, by Thermo Fisher Scientific (2 mentions)
Anti cd4 apc, by Thermo Fisher Scientific (2 mentions)
Anti cd11b pe, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Percoll gradient, by GE Healthcare (2 mentions)
Collagenase, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Live dead amine reactive violet viability marker, by Thermo Fisher Scientific (1 mentions)
3 protocols using percp cy5.5 anti cd8
1
Isolation and Characterization of Brain-Sequestered Cells
2
HCMV-specific T cell response assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMC or peptide epitope stimulated bulk T cell cultures were stimulated with either 1µg/mL of peptide or with IFN-γ activated HLA-matched fibroblasts infected with the HCMV strains TB40E or Merlin (kind gifts from Dr Barry Slobedman, Westmead Millennium Institute, Sydney, Australia) to assess endogenous antigen recognition. Cells were incubated for four to five hours in the presence of Brefeldin A (BD Biosciences), then incubated with PerCP-Cy5.5 anti-CD8 (eBioscience), fixed and permeabilised using a BD Cytofix/Cytoperm kit, and incubated with PE or AF700 anti-IFN-γ (BD Biosciences). Cell acquisition was performed using a FACS Canto II (BD Biosciences). Post-acquisition analysis was performed using FlowJo software (TreeStar).
3
Isolation and Flow Cytometry of Brain Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using FlowJo version 9.6.2. Mice were perfused for the analysis of brain sequestered cells. Brains were digested in RPMI containing 1.6mg/mL collagenase (type IV; Sigma-Aldrich) and 200μg/mL DNase I (Sigma-Aldrich) at 37°C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare) and debris was filtered out using a 70μm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability marker according to the manufacturer’s protocol (Invitrogen). The cells were blocked and labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), APC anti-CD4 (eBioscience; RM4-5) and PerCP-Cy5.5 anti-CD8 (eBioscience; 53–6.7).
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!