Delivery of membrane impermeable molecules to primary mouse T lymphocytes

STAR Protoc. 2021 Aug 17;2(3):100757. doi: 10.1016/j.xpro.2021.100757. eCollection 2021 Sep 17.


The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.

Keywords: Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Metabolism; Microscopy; Signal Transduction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / administration & dosage
  • Adenosine Triphosphate / pharmacokinetics
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / pharmacokinetics
  • Cell Membrane Permeability / drug effects*
  • Cell Separation
  • Drug Delivery Systems / methods*
  • Fluorescent Antibody Technique
  • Immunoblotting
  • Lymphocyte Activation
  • Mice
  • Molecular Biology / instrumentation
  • Molecular Biology / methods*
  • Receptors, Antigen, T-Cell / metabolism
  • Spleen / cytology
  • Streptolysins / chemistry
  • Streptolysins / pharmacokinetics*
  • T-Lymphocytes / drug effects*
  • T-Lymphocytes / metabolism


  • Bacterial Proteins
  • Receptors, Antigen, T-Cell
  • Streptolysins
  • streptolysin O
  • Adenosine Triphosphate