Measuring Leukocyte Migration to Nucleotides

Abstract

Extracellular nucleotides are potent damage-associated molecular patterns that shape the immune response to cell stress and tissue damage. These nucleotides are sensed by purinergic receptors and mediate a wide range of cellular effects. Among the best characterized of these effects is cellular migration. While the motility responses of leukocytes to nucleotides can be achieved by microscopic live-cell imaging approaches, such systems are time-consuming and require costly equipment and analysis tools not readily available to all researchers. Transwell migration chambers are a widely used alternative to microscopy due to their relatively low cost and moderate through-put capacity. However, extracellular nucleotides are labile and rapidly degraded in serum-containing cell cultures due to the presence of phosphohydrolases. Thus, evaluating leukocyte migration to nucleotides presents a number of challenges not seen with more stable classes of chemoattractants like proteins and lipids. Here we describe a method to measure leukocyte migration to nucleotides that is cost-effective, rapid and produces robust and reproducible migration of leukocytes using transwell migration chambers.

1. Introduction

Transwell chambers, also known as modified Boyden chambers, are one of the most commonly used systems to study the signaling processes that control cell migration to chemoattractants [1]. These assays are relatively simple to set up and, due to the availability of disposable transwell plates from a number of commercial vendors, are one of the first-line techniques used to study cell migration under very controlled experimental conditions. Conceptually, this is a fairly straightforward experimental approach. Chemoattractant-containing media is placed in the lower well of a tissue culture plate followed by addition of a removable upper chamber containing the cells interest in medium without chemoattractant. The lower portion, or “floor,” of the upper chamber is comprised of a synthetic (typically polycarbonate) mesh or “filter” manufactured to contain a discrete number of regularly spaced circular pores of defined diameter. The presence of chemoattractant in the lower but not upper chamber establishes a chemoattractant gradient at the interface of the upper and lower chambers that acts to stimulate chemotaxis. The assembled chamber is then incubated for a specific time and under controlled environmental conditions determined by the user necessary for sufficient chemotaxis to take place. Cells that traverse from the upper chamber through the filter and into the lower chamber are quantified using a variety of basic cell counting techniques, and the data are typically expressed as the fraction of cells originally placed in the upper chamber that have successfully migrated to the lower chamber (i.e., “percent migration”).

As migration through the transwell pores is dependent on establishment of a chemoattractant gradient, the stability of such chemoattractants is a major, if often underappreciated, factor in these assays. This is particularly true for migration induced by ATP and UTP. These nucleotides can have potent and rapid effects on leukocyte motility and have been shown to be important endogenous signals that enable swift response of immune cells to cellular damage in many contexts, including apoptotic cell clearance, antitumor immunity, and neuronal cell death [2-6]. However, extracellular triphosphate and diphosphate nucleotides are rapidly hydrolyzed to relatively inert monophosphates through the actions of phosphohydrolases present in serum-containing media as well as ectohydrolases expressed on the surface of migrating cells used in the assay. Chief among these ectoenzymes is CD39, whose expression has been shown to modulate cell motility and immune responses of many different leukocyte subsets [7]. However, as discussed below, we and others have successfully modified classical transwell chamber assay methods that account for nucleotide degradation in order to assess leukocyte migration to these important signaling molecules.

2. Materials

All materials should be prepared using sterile, tissue culture grade reagents and aseptic techniques throughout the assay to prevent microbial contamination of cultures.

2.1. Cell Culture

1. Here we describe transwell assays using the human monocytic cell line THP-1 as our exemplar cell type. For other cell types of interest, there are a number of important considerations and potential preliminary experiments required in choosing a cell type that will provide robust transwell migration results (see Note 1).

2. THP-1 growth medium: RPMI 1640 supplemented with 1× final penicillin/streptomycin/L-glutamine, 10% heatinactivated fetal bovine serum, 10 mM HEPES.

2.2. Transwell Migration Materials

1. Migration medium: RPMI 1640 supplemented with 1× final penicillin/streptomycin/L-glutamine, 0.5% bovine serum albumin, 10 mM HEPES. Once components are mixed and dissolved, filter through 0.2 μm filter top bottle and stored at 4 °C (see Note 2).

2. Transwell chambers: choose the appropriate plate size and filter pore size for your cell type (see Note 3).

3. Nucleotides: 1–1000 μM purified nucleotides dissolved in H₂O, stored at −20 °C.

2.3. Materials for Quantifying Migration

1. Flow cytometry quantitation beads: AccuCount Blank Particles (5.0 μm diameter).

2. Flow cytometer.

3. Methods

3.1. Cell Culture

1. THP-1 cells should be routinely maintained at a density of 0.2–0.5 × 10⁶/mL.

2. Approximately 16–24 h prior to the transwell experiment, pellet the cells by centrifugation at 350 × g for 5 min.

3. Resuspend cells in fresh THP-1 growth medium at 0.3 × 10⁶ cells/mL.

3.2. Transwell Chamber Setup

1. Thaw nucleotides on ice.

2. Dilute nucleotides in migration medium at desired concentration, typically from 0.1 to 1 μM.

3. With upper chamber in the well, load chemoattractant into the lower chamber for each condition (see Notes 4-6).

4. Load duplicate input control wells with 300 μL migration medium and no transwell insert.

5. Place plate in environmentally controlled incubator to warm and equilibrate for at least 30 min (see Note 7).

6. Collect cells by centrifugation, wash once in migration medium and resuspend in migration medium at 1 × 10⁶ cells/mL.

7. Load cells into the upper chambers of duplicate or triplicate transwells and also into input control wells containing same volume of medium as in lower chambers of transwells less the volume of input cells (see Note 8).

8. Place the plates in the incubator at 37 °C and 5% CO₂.

9. Incubate for 3 h.

10. Remove plates and carefully remove the inserts (see Note 9)—discard inserts.

11. Add 50 μL of flow cytometry counting beads per well.

12. Pipette medium in the well gently and transfer to flow cytometry tubes (see Note 10).

13. Record events on the flow cytometer by gating on beads and collecting a fixed number of bead events per sample (typically 2000–5000 bead events per sample). The number of cell gate events will allow you to calculate a relative migration amount compared to input controls.

14. Calculate the percent migration (see Note 11).