A single cell suspension from thymi and spleens of 4–6 week-old mice was obtained using a Dounce homogenizer. Cells were first preincubated for 10 min on ice with Fc receptor-blocking anti-CD16/32 (clone 93) antibody in PBS-1% biotin-free BSA solution and then stained for 20 min with specific primary antibodies. Biotinylated primary antibodies were revealed with streptavidin conjugated fluorescent dyes (SAv-PE/Cy7, SAv-APC, SAv-APC/Cy7). Labeled cells were analyzed on BD FACSCantoII (BD Biosciences) or CytoFLEX (Beckman Coulter) flow cytometers and data analysis was performed using FlowJo software (FlowJo, LLC). Clones of monoclonal antibodies were: anti-CCR6 (29–2L17), anti-CD3ε (145–2C11), anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.7), anti-CD11c (N418), CD24 (M1/69), anti-CD25 (PC61), anti-CD27 (LG.3A10), anti-CD28 (E18), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 (RI7217), anti-CD73 (TY/11.8), anti-CD117 (c-Kit, 2B8) anti-B220 (RA3–6B2), anti-CD11b (Mac1, M1/70), anti-γδT-cell (GL3), hamster IgG-PE/Cy7 (HTK888), anti-Ly6G/Ly6C (GR1, RB6–8C5), anti-Nk1.1 (PK136), anti-Tcrβ (H57–597), anti-Tcrβ(Vα2) (B20.1), Ter-119 (all from Biolegend). Splenocytes were analyzed as previously described (18 (link)).
Protocol detail
Murine Immune Cell Profiling
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
BD FACSCantoII
CytoFLEX
FlowJo software
anti-CD71
anti-CD73
anti-CD117
anti-B220
anti-Ly6G/Ly6C
anti-Nk1.1 (
anti-Tcrβ
Antibodies
Antibody
Biotin 1
C kit
Ccr6
Cd11b
Cd25
Cd3ε
Cd44
Cd71
Cd73
Cell
Clones
Fc receptor
Fluorescent dyes
Hamster
Mice
Monoclonal antibodies
Streptavidin
Tcrβ
Thymi
Corresponding Organization : Charles University
Other organizations : Czech Academy of Sciences, Institute of Experimental Medicine, Czech Academy of Sciences, Institute of Molecular Genetics, Albert Einstein College of Medicine
Top 5 similar protocols
Variable analysis
independent variables
- Preincubation of cells with Fc receptor-blocking anti-CD16/32 (clone 93) antibody in PBS-1% biotin-free BSA solution
- Staining of cells with specific primary antibodies for 20 min
- Expression of cell surface markers on the labeled cells
- Cell populations identified by flow cytometry analysis
- Cell source: 4-6 week-old mice (thymi and spleens)
- Cell preparation method: Dounce homogenizer to obtain single cell suspension
- Flow cytometry platforms: BD FACSCantoII and CytoFLEX
- Data analysis software: FlowJo
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!