Antibodies for cell surface staining for CD4 (RM4–5), D011.10 TCR (KJ1–26), CD25 (eBio3C7), CD44 (IM7), CD62L (MEL-14), GITR (YGITR 765), Ly6G (1A8), Ly6C (HK1.4), CD45, B220, MHC Class II (M5/114.15.2), CD11b (M1/70), CD3 (2C11), Siglec-F (E50-2440) and CD11c (N418) were purchased from eBioscience, Biolegend or BD Biosciences. Unlabeled anti-CD3 (2C11) and anti-CD28 (PV-1) were purchased from the University of California San Francisco Antibody Core. Intracellular staining for Foxp3 (FJK-16s), T-bet (4B10), IFNγ (XMG1.2), IL-17A (TC11-18H10.1), IL-10 (JES5-16E3), Ki-67 (B56) (eBioscience, Biolegend or BD Biosciences) was conducted using eBioscience Foxp3 intracellular staining reagents according to manufacturer’s instructions. Intracellular staining for phospho-STAT1 (4a) (BD Biosciences), phospho-SMAD2/3 (D27F4), phospho-AKT (D9E) and phospho-S6 Ribosomal protein (D57.2.2E) (Cell Signaling Technology) was conducted as previously described48 (link). Naïve CD4+ T cells were isolated using no-touch magnetic bead purification (MACS, Miltenyi Biotec). Where indicated, cells were further sorted as described by florescent antibody labeling using FACSAria cell sorting system (BD Biosciences). Flow cytometric analysis utilized LSRII, Canto, and FACSCalibur cytometers (BD Biosciences), and data was analyzed with FlowJo Software.
Protocol detail
Comprehensive 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.
Corresponding Organization :
Other organizations : King's College London
Top 5 similar protocols
Variable analysis
independent variables
- Anti-CD3 (2C11)
- Anti-CD28 (PV-1)
- CD4 (RM4-5)
- D011.10 TCR (KJ1-26)
- CD25 (eBio3C7)
- CD44 (IM7)
- CD62L (MEL-14)
- GITR (YGITR 765)
- Ly6G (1A8)
- Ly6C (HK1.4)
- MHC Class II (M5/114.15.2)
- CD11b (M1/70)
- CD3 (2C11)
- Siglec-F (E50-2440)
- CD11c (N418)
- Foxp3 (FJK-16s)
- T-bet (4B10)
- IFNγ (XMG1.2)
- IL-17A (TC11-18H10.1)
- IL-10 (JES5-16E3)
- Ki-67 (B56)
- Phospho-STAT1 (4a)
- Phospho-SMAD2/3 (D27F4)
- Phospho-AKT (D9E)
- Phospho-S6 Ribosomal protein (D57.2.2E)
- Naïve CD4+ T cells were isolated using no-touch magnetic bead purification (MACS, Miltenyi Biotec)
- Where indicated, cells were further sorted as described by florescent antibody labeling using FACSAria cell sorting system (BD Biosciences)
- Flow cytometric analysis utilized LSRII, Canto, and FACSCalibur cytometers (BD Biosciences)
- Unlabeled anti-CD3 (2C11) and anti-CD28 (PV-1) were purchased from the University of California San Francisco Antibody Core
- None specified
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!