RNA Isolation from Sorted Cells

RNA was isolated from fixed sorted cells based on the MARIS (method for analyzing RNA following intracellular sorting) protocol, as described by Hrvatin et al. (58 (link)). Briefly, we used FACS to obtain highly purified populations of T-CTLs, D-CTLs, M-CTLs, and N-CTLs from donors based on staining with CD3, GZMB, PRF, and GNLY, as described above. Before sorting, cells were fixed in 2% electron microscopy–grade paraformaldehyde (Electron Microscopy Sciences) and permeabilized with 0.5% deoxyribonuclease (DNase)/ribonuclease (RNase)–free saponin (Sigma) to permit intracellular staining. All staining and sorting took place in DNase/RNase-free phosphate-buffered saline (PBS) supplemented with microbiology-grade bovine serum albumin (Gemini Bio-Products) in the constant presence of RNasin plus RNase inhibitor (Promega) 1:25 to 1:100 (1:100 for washes and 1:25 for staining and sorting). After sorting, RNA was isolated using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion), as per the manufacturer’s instructions, with the same modification to the protocol used as described by Hrvatin et al. (58 (link)).

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.

Balin S.J., Pellegrini M., Klechevsky E., Won S.T., Weiss D.I., Choi A.W., Hakimian J., Lu J., Ochoa M.T., Bloom B.R., Lanier L.L., Stenger S, & Modlin R.L. (2018). Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria. Science immunology, 3(26), eaat7668.

Publication 2018

electron microscopy–grade paraformaldehyde RNasin plus RNase inhibitor RecoverAll Total Nucleic Acid Isolation Kit

Bovine serum albumin Cells Ctls Deoxyribonuclease Donors Electron microscopy Gzmb Intracellular Isolation Nucleic acid Paraformaldehyde Phosphate Populations Promega Ribonuclease Ribonuclease 5 Saline Saponin

Corresponding Organization : University of California, Los Angeles

Other organizations : Washington University in St. Louis, University of Southern California, Harvard University, Parker Institute for Cancer Immunotherapy, University of California, San Francisco, University Hospital Ulm

Top 5 similar protocols

Variable analysis

independent variables

FACS to obtain highly purified populations of T-CTLs, D-CTLs, M-CTLs, and N-CTLs from donors based on staining with CD3, GZMB, PRF, and GNLY

dependent variables

RNA isolated from the fixed sorted cells

control variables

Cells were fixed in 2% electron microscopy–grade paraformaldehyde and permeabilized with 0.5% deoxyribonuclease (DNase)/ribonuclease (RNase)–free saponin to permit intracellular staining
All staining and sorting took place in DNase/RNase-free phosphate-buffered saline (PBS) supplemented with microbiology-grade bovine serum albumin in the constant presence of RNasin plus RNase inhibitor
RNA was isolated using the RecoverAll Total Nucleic Acid Isolation Kit with the same modification to the protocol used as described by Hrvatin et al.

positive controls

Not explicitly mentioned

negative controls

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.

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?

Revolutionizing how scientists
search and build protocols!