A simple and rapid slide blot method to quantify cytokine-mRNA in small numbers of lymphocytes

Abstract

In this study we describe an in situ hybridization protocol suitable for the measurement of interleukin mRNAs in small numbers of cells in suspension. In this procedure defined numbers of such cells are coated onto defined areas of microscopic slides using LAB-TEK tissue culture chamber slides. 32P-labelled oligonucleotides are then hybridized to them in situ. Binding of the probes is measured by autoradiography using X ray film followed by densitometry (slide blot) or, as in standard in situ protocols, by exposure to photoemulsion. In experiments designed to demonstrate the specificity and feasibility of this protocol, peripheral blood T cells were activated with mitogenic stimuli and oligonucleotide probes specific for IL-2, IFN-γ, or a control probe (IL-2 random) were hybridized to them. Both lymphokine genes were found to be transcribed in two discrete phases with the first peak at 12 h and another peak 48 h after stimulation, as measured semi-quantitatively on a per cell basis using X ray film exposure. The proposed experimental protocol supports the advantages of X ray film autoradiography such as rapidity, simplicity and the objectivity of densitometric evaluation, but also permits microscopic evaluation of individual cells as performed in classical in situ hybridization protocols using photoemulsion.