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

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 10×10⁶ cells per 100 µl PBS. Keep on ice until i.v. injection. NOTE: cells have to be i.v. injected as quickly as possible. 6-12×10⁶ 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 (10×10⁶ 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.