Single-cell suspensions were prepared from liver, spleen, bone marrow, uterus, salivary gland, thymus, and lung tissues. Liver lymphocytes were isolated at the interphase of a 40/60% Percoll gradient (1 (link)). Uterus and salivary gland were digested in the presence of DNase I and collagenase D or liberase TL (5 (link), 7 (link)), while lung digestion additionally incorporated trypsin inhibitor. Cells were stained with a LIVE/DEAD fixable dead cell stain kit (Invitrogen) and blocked with antibodies against CD16/CD32 (BD) prior to staining with antibodies for CD3, CD11b, CD27, CD43, CD122, human CD5, integrin αv, KLRG1, Ly49G2, Ly49H, NKp46, and TRAIL (eBioscience); CD19, CD49a, CD49b, CD127, Ly49D, Ly-6G/Ly-6C (Gr-1), NK1.1, NKG2A/C/E, and TER-119 (BD). Cells were treated with cytofix/cytoperm kits prior to staining with antibodies for the transcription factors T-bet (eBioscience) and Eomes (eBioscience) or the cytokine TNF-α (BD).
Protocol detail
Comprehensive Isolation and Characterization of Diverse Immune Cell Populations
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.
Antibodies
Bone marrow
Cd11b
Cd122
Cd43
Cells
Collagenase
Cytokine
Digestion
Dnase i
Human
Integrin αv
Interphase
Klrg1
Liberase
Liver
Lung
Lymphocytes
Nkp46
Percoll
Salivary gland
Spleen
Stain
Thymus
Tissues
Tnf α
Trail
Transcription factors t
Trypsin inhibitor
Uterus
Corresponding Organization :
Other organizations : Columbia University, Aix-Marseille Université, Centre National de la Recherche Scientifique, Assistance Publique Hôpitaux de Marseille, Inserm, Centre d’Immunologie de Marseille-Luminy
Top 5 similar protocols
Variable analysis
independent variables
- Tissue type (liver, spleen, bone marrow, uterus, salivary gland, thymus, lung)
- Digestion method (with DNase I and collagenase D or liberase TL for uterus and salivary gland, with trypsin inhibitor for lung)
- Lymphocyte isolation (at the interphase of a 40/60% Percoll gradient for liver)
- Cell staining with various antibodies (CD3, CD11b, CD27, CD43, CD122, human CD5, integrin αv, KLRG1, Ly49G2, Ly49H, NKp46, TRAIL, CD19, CD49a, CD49b, CD127, Ly49D, Ly-6G/Ly-6C (Gr-1), NK1.1, NKG2A/C/E, TER-119, T-bet, Eomes, TNF-α)
- CD16/CD32 blocking prior to cell staining
- Positive control: Not specified
- Negative control: Not 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!