[Historical Background] In 1988, Andersson, et al. first demonstrated how lymphocytes could be fixed, permeabilized, stained with antibodies against IFNg, then fluorescently labeled and enumerated by flow cytometry. In 1991, Sander et al. demonstrated improved methods to fix cells with paraformaldehyde, permeabilize them with saponin, then use fluorescently-labeled antibodies to stain intracellular cytokines for microscopic examination. In 1993, Jung et al. extended this method to use with flow cytometry, and included the use of monensin to disrupt secretion, so as to increase intracellular signal of molecules otherwise released soon after synthesis. In 1995, Prussin and Metcalfe used directly-conjugated antibodies, and optimized the incubation period to 6 h. Also in 1995, Picker et al. considerably enhanced the sensitivity and reproducibility of cytokine detection by using Brefeldin A to block the secretion apparatus for cytokines, and by using a different permeant (Tween-20). In 1997, this matured method was applied by Picker et al. to study the the antigen-specific homeostatic mechanism in HIV⁺ patients. In 2001, Schuerwegh et al. confirmed that BfA provides for better cytokine signals than monensin, used by others in this method.

In two reports in 1989, one by Gardner et al. and the other by McClure et al. reported that Rhesus macaques were a useful model for studying HIV disease and AIDS. In 2002, Picker et al. reported the application of the CFC assay to Rhesus macaques. In 2012, a group created to develop multi-lab standards for use of ICS in NHP vaccine studies published their recommendations for a 96-well plate method with a 6 h total incubation (Donaldson et al., 2012 and Foulds et al., 2012).

The general procedure reported here is that 2002 tube-format method, now with a 9 h total incubation, and optimized especially for low-end sensitivity. The specific details here are the state of the art now practiced by the Louis Picker Lab, at the Oregon Health and Science University, affiliated with the Oregon National Primate Research Center. These methods have been used in several of our recent publications (Fukazawa et al., 2012; Hansen et al., 2011; Hansen et al., 2009). It is important to note that in our hands, plate-format ICS is not as reliable or sensitive for weak responses as is this tube-based method (unpublished observations). Until that problem is understood and solved, the tube-based method remains the most-sensitive format for CFC.

Materials and Reagents

Data required: Flow cytometry data files, from CFC assay samples as described in “From Assay Set-up to Data Acquisition” (Sylwester et al., 2014).

Equipment

Incubator for tissue culture (humidified, stable at 37 °C, 5% CO₂ atmosphere)
Flow cytometry analyser

Software

Software-equipped workstation, for analysis of cytometry files
e.g. FlowJo (Tree Star)
e.g. SPICE (NIAID)

Procedure