Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry.

Abstract

Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.

Measurement of Intracellular Ions by Flow Cytometry

“…However, if cellular Indo-1 loading is extremely heterogeneous, convert violet and blue emission intensities to log values in order to observe a broader range of cellular fluorescence. In this case, the logarithm of the ratio is calculated by subtracting the log of the blue signal from the log of the violet signal (Rabinovitch et al, 1986).…”

“…In addition, the ratio of Indo-1 signals allows measurement of [Ca 2+ ] i that is independent of cell size or brightness. Cells loaded with Indo-1 exhibit a 3-fold range in brightness, and after measuring the ratio of Indo-1 calcium-bound to calcium-unbound at the proper wavelengths, the cell-to-cell variation in the signal from resting cells has a coefficient of variation of only 5% to 10% (Rabinovitch et al, 1986). Thus, with Indo-1 it is possible to discriminate responses of small subpopulations of cells within a larger population of non-responding cells.…”

“…A correctly calculated ratio will not show dependence upon excitation intensity. Loading cells with a broad range of Indo-1 or Fluo-3 and Fura Red concentrations should also result in a constant value for the violet:blue or Fluo-3:Fura Red ratios (Rabinovitch et al, 1986;Novak and Rabinovitch, 1994).…”

“…If the cells are too dim, excessively bright, or if the dye is compartmentalized, the ability to detect calcium signals will be impaired. For unknown reasons, the calcium signaling of B cells and not T cells is particularly sensitive to overloading with Indo-1 (Rabinovitch et al, 1986;Chused et al, 1987). The cells must be suspended in medium that contains calcium; without it, responses are blunted, in for example, PBS without added calcium or magnesium.…”

“…Alternative calibration approaches have been proposed. Determining R max , R min , and S f2 / S b2 for calibration of Indo‐1 ratios allows the direct determination of the constants in the equation that relates Indo‐1 fluorescence to [Ca 2+ ] i (Rabinovitch et al., ). A novel method employing measurements of spectral shifts has also been proposed for calcium calibration (Kachel et al., ).…”