Light scatter properties of cultured murine macrophage and human T cells

Light scatter properties of cultured murine macrophage and human T cells.

Introduction
Flow cytometry has been a cornerstone in biological advancement, and in immunology in particular over the last 30 years. This technique achieves high levels of statistical robustness via high throughput analysis of individual cells using lasers, light scatter and fluorescent markers. Light scatter is a crucial component of flow cytometry that uses forward scatter to discern relative size and side scatter to measure relative internal complexity of individual cells. These properties can be used to gate out doublets, clumps and debris, identify distinct cell populations, or to look at any relative morphological changes within a population such as activation. Here in we will analyse the light scatter properties of Jurkat T cells and RAW macrophage cells using flow cytometry to understand how we can employ side and forward scatter to produce the best possible data in our future experiments.

Materials and Methods

Forward and side scatter analysis of in vitro cultured cell lines
RAW murine macrophage and Jurkat T lymphocytes were processed following the MMI 590 Lab 1 protocol to be run on a BD FACS Canto II flow cytometer. Briefly, cultured cells were spun down and resuspended in FACS Buffer. Cells were counted and due to low cell concentrations, 1.6x10⁵ RAW cells and 5.7x10⁵ Jurkat cells (in 1mL) were seeded into their respective FACS tube. The combined tube held  7.9x10⁴ and 2.9x10⁵. Combined results used in the data portion were obtained from Group 2 due to long sample acquisition times from low cell concentrations. FACS Canto II lasers were set at 330mV and 450mV for FSC and SSC respectively.

Results and Discussion

Light scatter can be employed to remove debris and doubletsCellular light scatter properties are an important tool when setting up any good flow cytometry experiment, as it can help to remove events that may skew results and thus the conclusions garnished from the experiment. The gating strategy used in this experiment (Figure 1a) displays this importance with 11.8% of total events (black dots) gated out in the P1 gate which is mostly debris of low size (forward scatter). The population remaining contains both the RAW and Jurkat cell populations within them, but there are still likely clumps and doublets that may skew data, especially in experiments looking for co-expression or other double-positive events since two single positive cells may appear as a double positive event if not gated out properly. To achieve this, cells with a low ratio of forward scatter area vs height are gated out (P2, red dots). When two cells are counted as a single event, their area to height ratio will be roughly twice that of a single cell event pulling the respective dot out of the bottom of the P2 gate and disregarding it from the dataset. In the mixed population, this removes an additional 12.4% of total events captured for a total of 24.2% of events removed with 52.8% and 20.7% of events removed for RAW and Jurkat only samples respectively (Figure 1b). Removing these events makes the dataset and thus and conclusions made more robust.

Figure 1. Light scatter profiles of RAW and Jurkat cell lines. a) Gating strategies on a mixed RAW and Jurkat cell population included excluding low side scatter and forward scatter events (P1 red and green). High FSC area to FSC height ratio events (red) were then excluded from this population to yield a population for final analysis (green). b) Forward and side scatter plots for post-gated RAW macrophage, Jurkat T- lymphocytes and a mixed population containing both cultures. Proportion of total captured events remaining for analysis is indicated at the top right of each plot.

Light scatter can be used to distinguish distinct cell populations
Light scatter can also be used to distinguish cell populations that are morphologically different by relative size measured by forward scatter, or relative internal complexity measured by side scatter.
We saw that the RAW macrophage sat, on average, at a lower forward scatter than the Jurkat cells indicating that they are on average smaller, although the Jurkat only population had proportion of cells that sat at the smaller range of RAW cells (figure 1b). These could be apoptotic cells and could possibly be gated out by a viability assay. Unfortunately, the use of some kind of fluorescent marker would be needed to separate these two cell populations in flow analysis because of their overlapping forward and side scatter profiles.