Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Summary

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Abstract

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.

Here we present a protocol for imaging monocytes and neutrophils in the CX₃CR1^gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.

The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

Introduction

Neutrophils and monocytes are cells of the innate immune system that continuously circulate in the blood. Upon injury or infection, inflammatory signals induce leucocyte diapedesis into damaged and infected tissues, herein initiating a cellular immune response ^1-3. The rapidity of leukocyte mobilization determines the positive outcome of the immune responses. These intricate processes rely on specific molecules (e.g., selectins, endothelium-bound chemokines) present on the inflamed endothelium that help for the establishment of adhesive contacts between circulating leukocytes and the endothelium ^1-3.To get insights on the molecules implicated in the leukocyte recruitment cascade, it is important to visualize the kinetics of cell recruitment and to track the behavior of each cell/population. An effective method involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.

To date, several approaches using intravital microscopy were developed to image the vasculature ^4,5. For instance, imaging of ear dermis vasculature or mesenteric veins by intravital confocal microscopy was used to identify the patrolling behavior of murine Ly6C^low monocytes and human CD14^dimCD16⁺ monocytes on the luminal side of blood vessels under steady state conditions ^6-8. The mouse cremaster vasculature model is often used to monitor the behavior of neutrophils or inflammatory Ly6C^high monocytes under inflammatory or ischemic conditions in transgenic mice. In that case, cremaster is stimulated via intrascrotal injection of IL1β, CCL2, TNFβ or fMLP. After 2-4 hr, tissues are then surgically exteriorized and analyzed by intravital confocal microscopy ^9-11.

The following protocol describes a method to monitor monocytes and neutrophils at the same time with any inverted fluorescence confocal microscope. Furthermore, our method details how to image the same vessel before (steady state condition) and after inflammation and how to follow the kinetics of leukocyte recruitment. To this purpose, we use the CX₃CR1^gfp/wt mouse, whose monocytes express eGFP, i.v. injected with an orange dye-labeled murine neutrophils. Using an inverted confocal microscope, it is possible (1) to track and analyze the patrolling Ly6C^low monocytes under steady state conditions and (2) to follow the recruitment of both monocytes and neutrophils in the same vessel after local inflammation. Here we create the inflammatory conditions by using the TLR2/TLR1 agonist Pam3CSK4 ¹². Moreover, such imaging may help to determine the role of specific molecules of interest in the various steps of the leukocyte recruitment cascade if performed on specific knock-out mice or in presence of blocking antibodies ^6,9,13.

Protocol

NOTE: Animal procedures were performed in accordance with the Institutional Ethical Committee of Animal Care in Geneva, Switzerland and the Cantonal Veterinary Office. Authorization number GE/63/14.

1. Preparation of a Single Cell Suspension from Bone Marrow

1. Sacrifice mouse (8-12 week old) by cervical dislocation. Sterilize hind legs with 70% ethanol. Remove skin from hind legs.
2. Dissect out mouse femurs and tibias and remove tissue from legs with a scalpel. Rinse legs with 90% ethanol and place into a culture dish filled with PBS.
3. Under the culture hood, clean tissue from femurs and tibias with a scalpel and separate at knee joint. Cut off each end of the bones.
4. Flush bone marrow from each bone using a 23G needle and a 1 ml syringe filled with preparation medium (phenol red-free DMEM/F12, 1% rat serum), into a 50 ml screw tube. Disrupt cell aggregates by delicately passaging through the 23G needle and a 1 ml syringe. Bring total volume to 25 ml with preparation medium.
5. Centrifuge 250 x g, 5 min, 4 °C. Discard supernatant. Resuspend pellet in 1 ml Red Blood Cell Lysis RBLC buffer (8.3 g NH₄Cl, 1 g NaHCO_3, 1 ml EDTA (100 mM), complete to 1 L with distilled water, filter 0.22 µm) in a 15 ml screw tube. Incubate 30 sec on ice.
6. Add 10 ml of preparation medium and centrifuge 250 x g, 5 min, 4 °C. Discard supernatant and resuspend in 2 ml preparation medium.

2. Neutrophil Enrichment by Negative Selection

1. Add 150 µl of Mouse neutrophil enrichment cocktail (cell isolation platform kit such as EasySep). Mix and incubate on ice for 10 min.
2. Add 10 ml of phenol red free DMEM. Invert once and centrifuge 250 x g, 3 min, 4 °C.
3. Discard supernatant. Resuspend pellet in 1.75 ml preparation medium in 5 ml polystyrene round bottom tubes. Add 250 µl Biotin Selection Cocktail. Mix and incubate on ice for 10 min.
4. Vortex the magnetic particles (from the cell isolation platform kit) for 30 sec to resuspend particles. Add 500 µl magnetic particles to cell suspension. Mix and incubate on ice for 10 min.
5. Place the tube into the magnet. Wait 3 min.
6. Invert the magnet onto a new 5 ml polystyrene round bottom tubes to collect the desired unlabeled cells. Do not take the last drop hanging in the tube. Unwanted cells will remain in the tube inside the magnet. Discard this tube into a biohazard waste.
7. Place the new tube into the magnet. Wait 3 min.
8. Invert the magnet onto a new 15 ml screw tube to collect the desired unlabeled cells. Do not take the last drop hanging in the tube.
9. Complete volume to 5 ml with phenol red free DMEM.

3. Labeling of Neutrophils

1. Add 0.5 µl of an orange dye such as Cell Tracker Orange (working concentration 1 µM, CMRA – chloromethyl-rodol-acetate, stock 10 mM in DMSO). Incubate 2 min at 37 °C.
2. Add 2.5 ml of preparation medium. Centrifuge 250 x g, 5 min, 4 °C.
3. Discard supernatant. Resuspend pellet in 1 ml preparation medium in a 1.5 ml tube.
4. Take an aliquot to count with hematocytometer.
5. Incubate 5 min at 37 °C. Centrifuge 250 x g, 5 min, 4 °C.
6. Discard supernatant. Resuspend pellet in 1 ml PBS. Centrifuge 250 x g, 5 min, 4 °C to wash.
7. Discard supernatant. Resuspend pellet as 10x10⁶ cells per 100 µl PBS. Keep on ice until i.v. injection. NOTE: cells have to be i.v. injected as quickly as possible. 6-12x10⁶ neutrophils/mouse are usually obtained.

4. Mouse Preparation for Intravital Microscopy

NOTE: Step 4.1.) and 4.2.) should be performed at the beginning of the experiment to avoid extended waiting time of labeled neutrophils on ice.

1. Weigh the CX₃CR1^gfp mouse (8-12 week old).
2. Prepare 800 µl of anesthetics (ketamine 60 mg/kg, xylazine 4.5 mg/kg, acepromazone 1.75 mg/kg in PBS).
3. Anesthetize mouse. Injection i.p. with 200 µl / 20 g of mouse. Control anesthesia by toe pinching and by checking the respiratory rate.
4. Inject neutrophils (10x10⁶ cells in 100 µl PBS) intravenously using the lateral tail veins or the retro-orbital sinus, at the convenience of the experimenter.
5. Put the mouse on heating pad and protect the eyes with ocular gel.
6. A glass coverslip (35 mm diameter) is put in a 10 cm tissue culture dish, which has a 30 mm diameter hole. Use oil is to maintain the tissue culture dish in contact with the coverslip.
7. Incise abdominal skin with scissors to expose the peritoneum. Incise peritoneum with scissors to expose intestine.
8. Add 200 µl PBS (pre-heated at 37 °C) into the peritoneal cavity. Take the mouse in your hand and turn it face down, so that the intestine gets out of the peritoneal cavity with a gentle pressure. Place the mouse in the tissue culture dish.
9. Gently deroll the intestine with sterilized cotton buds onto the coverslip in order to expose the mesenteric vessels.
10. Cut paper tissues in bands and wet them with 37°C heated PBS. Immobilize the intestine with the pieces of paper tissues to decrease the movements resulting from peristalsis.
11. Put the tissue culture dish and the mouse inside the 37 °C thermostated chamber onto the custom-made tray stage of the inverted microscope. Introduce a syringe containing anesthetics s.c. into the hind leg. NOTE: Additionally, the mouse can receive oxygen (0.5 L/min) with a mask.
12. Control mouse for anesthesia by toe pinching and checking the respiratory rate every 30 min. If needed, deliver a boost of anesthetics (20 µl) to properly maintain anesthesia. NOTE: Drying of the vasculature should be avoided. Therefore, its humidity is maintained by regularly wetting the paper tissues used to immobilize the intestine with 37°C-warmed PBS.

5. Measuring Neutrophil and Monocyte Behavior in Inflamed Vessels In Vivo using Intravital Microscopy

1. Set lasers to 488- and 561- at the lowest power necessary to avoid laser-induced phototoxicity. In our experiments, it is lower than 0.5%. Intensity of GFP in monocytes and orange dye in neutrophils is so bright that usually 0.3% is enough. NOTE: This represents a laser power of 0.15 to 0.25 mW with our system.
2. Acquire images of 512 x 512 pixels (i.e., 635 µm) in 2.2 sec with an open pinhole using the 20X dry objective of the inverted microscope. Use a z-depth between 10 to 20 μm, that ensures sufficient signal with the low laser powers.
   NOTE: We usually perform experiments with a z-depth between 12 to 16 μm. In the case of presented figures and movies, the z-depth is 20 μm. Phase contrast allows the identification of endothelial walls and imaging is performed on mesenteric veins. Since patrolling cells move quite slowly with a speed of 5-10 µm /min ⁶, recording a field of interest every 20 sec is satisfactory. With the described settings, the motorized stage allows the recording of up to 7 fields of interest at the same time.
3. Place the mouse and the mesenteric vessels in the thermostat chamber of the microscope are allow them to recover from the preparation for 30 min before the acquisition of the first movie. During this time, choose several fields of interest. Focus on the luminal surface of the vessel closest to the objective. If needed, take advantage of the slight auto-fluorescence of endothelium to set the focus.
   NOTE: Surgery during mouse preparation slightly stresses the endothelium. Consequently, rolling of neutrophils and/or monocytes can be observed in the first 15 min following mouse preparation.
4. Record a first movie for 30 min under steady state conditions, one image every 20 sec.
   NOTE: The software acquires each field of interest in 2.2 sec and the motorized stage automatically moves from one field to the other. It goes back to the first position within 20 sec during the 30 min of the recording movie.
5. To initiate blood vessel inflammation, add a drop of 20 µl of PBS containing 100 µg of TLR2/TLR1 agonist Pam3CSK4 ¹² directly onto the imaged vessels.
6. Control the focus for each field of interest.
   NOTE: During the course of acquisition, there is always slight changes of focus on z-axis. Despite the 10-20 μm z-depth, this can result in a decrease of fluorescence intensity. Therefore, manually refocus each field of interest at the end of each movie, if needed. Record movies for 30 min-periods and up to 3 hr total after initiation of inflammation.
7. At the end of the experiment, sacrifice anesthetized mouse by cervical dislocation.

6. Generation of Movie Files and Tracking of Leukocytes

NOTE: The Nikon acquisition software generates .nd2 files but the following procedure would be similar with other software and brands.

1. Export files as tiff series.
2. Go to ImageJ software (http://rsb.info.nih.gov/ij/). Import the tiff series. Each channel as a different file.
3. Apply a median filter with a radius of 2.0 pixel for the green and red channels to reduce background noise (Process > Filters > Median…).
4. Convert files into binary (Process > Binary > Make Binary). Enable the software to calculate the threshold for each image.
5. Merge channels into one image series (Image > Color > Merge Channels…). Select the monocytes in the green channel, the neutrophils in the red channel and the transmitted light in the grey channel.
6. Convert stacked images into RGB colors (Image > Color > Stack to RGB).
7. Save file as .avi
8. Data are imported and compiled in Imaris software (Bitplane). Movements of monocytes and neutrophils are then tracked using the Spot Tracking Wizard.

Results

The manuscript describes an optimized protocol to easily monitor the behavior of monocytes and neutrophils in the mesenteric veins of anaesthetized mice in real time. The use of a 37 °C-thermostated chamber is mandatory to maintain the temperature of the mouse and also due to the temperature dependent movement of leukocytes. Preparation of the mouse is displayed in Figure 1. Figure 2 shows all the area seen under the microscope. Transmitted light allows the identification of mesenteric veins (red ar...

Discussion

The methodologies described in this manuscript provide a consistent approach to efficiently study monocyte and neutrophil behavior in mesenteric veins under steady state and inflammatory conditions.

The crucial step of the preparation is the immobilization of the intestine with PBS-wetted tissues. If performed properly, mesenteric vessels are nicely exposed on the coverslip for image acquisition. This enables the selection of several fields of interest to monitor leukocyte behavior in mesenter...

Materials

Name	Company	Catalog Number	Comments
5 ml polystyrene round bottom tubes	Beckton Dickinson	352058
10x CFI Plan Apochromat 0.45   DT:4mm	Nikon
20x CFI Plan Apochromat VC 0.75 DT:1mm	Nikon
Cell Tracker Orange CMRA Dye	Life Technologies	C34551
EasySep Magnet	Stem Cell Technologies	18000
EasySep Mouse Neutrophil Enrichement kit	Stem Cell Technologies	19762
EDTA	Sigma Aldrich	E6758
FCS	PAA	A15-042
Immersion Oil Type A	Nikon		any viscous oil
Life Box Temperature Control System	Life Imaging
NaHCO3	Sigma Aldrich	S5761
NH4Cl	Sigma Aldrich	A9434
Nikon A1R confocal microscope	Nikon	A1R	inverted microscope, motorized x/y/z stage, NIS elements software
PBS	Life Technologies	D8537
phenol red free DMEM/F12	Life Technologies	21041-025	any phenol red free medium is suitable
PAM3CSK4	Invivogen	tlrl-pms	reconstitute in PBS
Rat serum	Stem Cell Technologies		included in EasySep Mouse Neutrophil Enrichement kit
Tissue culture dish 100	TPP	93100