Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Summary

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Abstract

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45⁺ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45⁺ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45⁺ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45⁺ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

Introduction

We describe here efficient and reliable methods of differentiating intravascular leukocytes from pulmonary leukocytes. Even with the best perfusion techniques, studies have revealed residual CD45⁺ from circulation persists in the lung. This impairs the ability to distinguish between the rhythms in the circulation and the lung. This effect is further amplified in cases of lung inflammation. This is particularly relevant for the study of circadian regulation of inflammation.

Circadian rhythms refer to the diurnal oscillations in various biological processes that occur with a period of 24 h. The circadian system is an evolutionarily....

Protocol

All animal studies were approved by the University of Pennsylvania Institutional Animal Care and Use Committee and met the stipulations of the Guide for the Care and Use of Laboratory Animals.

NOTE: The overall process may be divided into 1) intravenous CD45 labeling, 2) harvest, 3) digestion, and 4) staining and flow cytometry. These steps have been summarized in Figure 1.

1. Solutions/Reagent preparation

1. Prepare Dissociati.......

Discussion

Careful studies of lung inflammation and pulmonary immune responses are crucial to the understanding of many disease conditions. Flow cytometry is routinely used to enumerate and ascribe functional relevance to pulmonary leukocytes. The function of leukocytes depends at least partly on where they are found. Although there is accumulating evidence to support that even after perfect perfusion protocols, many intravascular leukocytes persist in the lungs, most studies do not differentiate between intrapulmonary and intravas.......

Materials

Name	Company	Catalog Number	Comments
Boekel Scientific Medium Water Bath	Boekel Grant Scientific	290200
10 mL BD Syringes with BD Luer-Lok Tip	BD Biosciences	309604
5 mL BD Syringes with BD Luer-Lok Tip	BD Biosciences	309646
Anti-CD45- Pac Blue	Biolegend	103114
Anti-CD45- Pe/Cy7	Biolegend	103114
Cell strainer 70 µm Nylon	Fisher	352350
Corning Conical-Bottom Centrifuge Tube 50 mL	Avantor	21008-714
Corning Falcon Test Tube with Cell Strainer Snap Cap	EMSCO	10004637
Dissection Microscope	Olympus	SZX-SDO2
DMEM, high glucose	Life Technologies	11965084
Dnase	Roche	10104159001
DPBS without Ca++ & Mg++		14190136
Fc Block	Biolegend	101320
HyClone Fetal Bovine Serum	GE Healthcare	SH30071.03
L-Glutamine (200 mM)	Life Technologies	25030-081
Liberase Research Grade	Sigma	5401127001
Penicillin-Streptomycin (10,000 U/mL)	Life Technologies	15140-122
Precision Shaking Water Bath	Thermo Fisher	TSSWB15
Red Blood Cell Lysing Buffer	Sigma	R7757
Suture Silk 4-0	Roboz	SUT-15-2