Two new protocols added to the Current Protocols in Cytometry:

  1. Measurement of autophagy by flow cytometry (Gary Warners): In recent years, flow cytometry has been used to detect the presence of autophagy mainly by the fluorescent antibody labeling of the autophagy marker LC3-II. Here we describe the use of the indirect antibody labeling of LC3-II as well as LysoTracker dyes to investigate the autophagic process induced by rapamycin, chloroquine, and serum starvation. This approach shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, LC3-II/LysoTracker allow the simultaneous measurement of an autophagy related process and other live cell functions, which is not possible with the standard LC3-II antibody technique.
  2. Immunophenotyping of paucicellular samples (A. Stacchini, A. Demurtas, and S. Aliberti): Immunophenotyping of paucicellular samples (<0.3 × 10^6 total cells) may represent a challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the numer of test that can be performed. In this protocol, an 8-color/10-antibody/13-parameter single-tube FC Assay, whcih includes the use of a cell viability dye, doublet discrimination, and a Boolean gating strategy, is used to identify leukocytes, quantify the main lymphocyte populations, detect lymphomatous B cells, or detect any aberrant T cell expression if present. Must to see!