Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Summary

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Abstract

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Introduction

Chemokines are small soluble proteins that attract cells to specialized locations¹. Therefore, they participate in the correct positioning of cells in tissues, a crucial function in development and physiology. The immune system is no exception to this rule as it relies on the action of many different cell types which act in concert to mount an effective immune response. By controlling the specific location of one immune cell type in a given state, chemokines are pre-required before foreign antigens can be detected and neutralized.

In T lymphocytes in particular, chemokines bind to specific surface receptors which, upon engagement....

Protocol

1. Preparation of T Lymphocytes

1. Prepare primary mouse or human T cells as described^10,11.
2. Cultivate human T cells in complete RPMI medium with 10% human serum AB at a density of 2 - 3 x 10⁶ cells/ml.
   NOTE: The use of fetal calf serum (FCS) instead of human serum can elicit spontaneous polarization of a large fraction of human T cells in culture. Keep these cells in culture for 4 - 5 days and further process them at any time during this period.
3. Maintain mouse T cells in complete RPMI medium supplemented with 10% FCS at a similar cell density. Use right away or the next day, after an O/N culture at 37 °C ....

Results

The first example chosen here concerns the use of human primary T lymphocytes stimulated with CCL19. However, the same strategy can be used with primary mouse T cells, T cell lines or any other cell type responsive to chemokine stimulation. The gating strategy presented here includes a series of windows that select the focused events, then the single cells. Finally the range of fluorescence intensity is followed here as an example on two markers: the MHC Class I HLA-ABC and filamentous actin for resting (Figure 1.......

Discussion

Using a recent technology of imaging flow cytometry, a rapid and informative gating strategy to analyze cellular and molecular events induced by chemokine stimulation is presented. From a single experiment, one can obtain two main types of information: the changes in cell morphology induced by chemokine stimulation and the subcellular distribution of different proteins during the polarization process. Interestingly, evidence is also provided for the possibility to analyze a large number of cells, which allows statistical.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI	Gibco	61870-010
Human serum AB	PAA	C11-021	Pre-heat to inactivate the complement.
Fetal calf serum	PAN Biotech	P30-3300	Pre-heat to inactivate the complement.
Human T cell nucleofactor kit	Lonza	VCA-1002
Murine IL7	Peprotech	217-17
HBSS	Gibco	14025-050	Warm in 37 °C water bath before use.
Hepes	Gibco	15630-056
Murine CCL19	Peprotech	250-27B	Aliquots are thawed on ice before adding the chemokine to the cells.
Human CCL19	Peprotech	300-29B	Aliquots are thawed on ice before adding the chemokine to the cells.
Human CXCL12	Peprotech	300-28A	Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.
PFA	Electron Microscopy Sciences	157-8-100	This is a 8% PFA solution in water. Mix volume to volume with 2x PBS to obtain a 4% PFA solution in PBS.
BSA	Sigma	A3059
Saponin	Fluka	84510
Alexa Fluor 594 phalloidin	Invitrogen	A12381
FITC-anti-HLA-ABC antibody	Beckman Coulter	IM1838	clone B9.12.1
Anti-P-ERM antibody	Cell Signaling Technology	3149P
ImageStreamx Mark II	Amnis