Multiplexed labeling of samples with cell tracking dyes facilitates rapid and accurate internally controlled calcium flux measurement by flow cytometry

Abstract

Calcium flux measurement is a crucial assay in lymphocyte activation. However, with the currently well established flow cytometric methods, it is a tedious procedure that is difficult to control to avoid variation between samples. This leads to unwanted sources of error that can make it problematic to interpret the results. Here we present an improved method that allows different cell populations to be tested in the same sample. Samples are pre-labeled with CFSE or Cy5 then mixed and stimulated to induce calcium flux. This facilitates more rapid and accurate measurement of calcium flux and also dramatically reduces the cost and effort required for this type of assay.

Fig. 1

Typical errors in standard calcium flux assay and the proposed new method. It is easy to introduce varations in calcium flux assay, shown here are two typical errors caused by either inaccurate timing of adding reagents (A), or inconsistent instrumental flow rate between samples (B). These situation prompted us to improve the current standard approach in order to eliminate these varations. The difference between standard and alternative approaches is shown in (C).

Fig. 2

Development of the improved calcium flux method. CFSE and Indo-1 labeling are compatible: (A) Sequential labeling of cells with Indo-1 does not affect previous CFSE labeling as determined by FACS. (B) Conversely, CFSE labeling does not interfere with Indo-1 labeling reflected by normal cellular calcium flux. Shown is the calcium flux kinetics of CD8⁺ lymph node T cells. Gray, CFSE labeled population; Black, mock labeled population. Validation of the improved method: (C) In control sample, there was no detectable difference in calcium flux between CFSE-labeled, Cy5-labeled, and mock-labeled subpopulations. (D, E, F) In test samples mixed in a “rotated” manner, both Itk^−/− and Coro1a^Lmb3 cells showed a calcium flux defect compared to wild type cells.

Fig. 3

Application of the improved calcium flux method to PKCη-deficient thymocytes. In a real application of the improved method, CD4⁺CD8⁺ PKCη^−/− thymocytes showed no defect in calcium flux. The experimental design and data analysis models are depicted in (A), whereas a, b, c, and d are four proposed analysis models. (B) The calcium flux results are analyzed in the manner according to a, b, c, d in (A).