In vitro Monitoring of Extracellular pH in Real-Time

Summary

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Abstract

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H₂O₂, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

Introduction

The extracellular microenvironment has been shown to play a significant role in modulating the inflammatory response. One aspect of the microenvironment which is often underappreciated is extracellular acidification. Extracellular acidification is often observed at sites of active inflammation, including mucosal disorders such as inflammatory bowel diseases. The luminal pH in the distal colon from patients with UC can range between a pH of 5 and 6, whereas healthy individuals have colonic pH's in the range of 6.8-7.4^1,2. This decrease in colonic pH is of particular interest because extracellular pH has been sh....

Protocol

1. Cell Preparation

Day 1

1. Warm cell culture media (DMEM/F12 with 5% FBS, 2 mM L-alanyl-L-glutamine dipeptide, and Pen Strep) in a 37 °C water bath for 20 min.
2. Prepare the tissue culture hood by spraying it with 70% ethanol and wiping down the surfaces with paper towels.
3. Spray and wipe tissue culture flasks and media, PBS, and trypsin bottles with 70% ethanol and bring them into the tissue culture hood.
4. Aspirate the media from the cell culture flask and wash the cells with 12 mL of phosphate buffered saline (PBS).
5. Aspirate the PBS and add 1 mL of trypsin to the cells. Incubate at RT fo....

Results

The results are usually via line graph to show change in pH over time (example shown in Figure 3A) or as a scatter plot showing extracellular pH at a single point in time (example shown in Figure 3B). Depending on experimental need, additional controls and treatments can be included. Additionally, this assay can be modified to monitor extracellular under a wide range of conditions. For example, the SDR reader can be placed in a hypoxic chamber and the extracellu.......

Discussion

In this protocol, there are several key steps. Monolayers should be confluent, but not overconfluent. For T84 IEC, they should be used 7-10 days after plating. Human and murine enteroids grow at different rates than T84 IEC and the researchers should determine how long it takes each line to reach confluency. It is important that the researchers need to use minimally buffered media to ensure shifts in extracellular pH are observed. HBSS+ contains glucose and is suitable for experiments shorter than 12 h. T84 IEC incubated.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL serological pipettes	Corning	4101
24-well plate	Corning	CLS3527-100EA
5 mm pore inserts	Corning	3421
50 ml sterile conical tube	Corning	0553855A
75 cm2 flask	Corning	430641U
DMEM/F12	Gibco	10565-018
FBS	Gibco	26140
GlutaMax	ThermoFisher	35050061
HBSS-	Sigma-Aldrich	H4891-10X1L
HBSS+	Sigma-Aldrich	H1387-10L
Histopaque T1077	Sigma-Aldrich	10771-6X100ML
Histopaque T1119	Sigma-Aldrich	11191-6X100ML
HydroDish HD24	PreSens	NA	https://www.presens.de/products/detail/hydrodish-hd24
PBS	Gibco	14190-144
Pen Strep	Gibco	15140-122
RBC lysis buffer	ThermoFisher	00-4333-57
SDR Reader	PreSens	NA	https://www.presens.de/products/detail/sdr-sensordish-reader-basic-set
Trypsin	Fisher Scientific	25200114