2-Photon Imaging of Ex-Vivo Lymph Node By Yi-Chun Maria Chen and Jessica Wong
Excised lymph nodes are a good sample for testing depth penetration. Lymphocytes of a transgenic mouse can be adoptively transferred into a wild type mouse so that only a small population of fluorescent cells are present for imaging. This protocol was written for the Matthew Krummel's 2-photon section of CSHL's Quantitative Imaging Course.
Tools and Materials
- Cell Strainer
- 5ml Syringe
- 5 cm dish
- 6 well dish
- Plastic coverslip
- RPMI-10 (RPMI +FCS +PSG +Bme
- RPMI (no phenol red)
- Vacuum Grease
- Dissection Tools
- Dissection Microscope
- Heat Lamp
- Heated Perfusion Chamber
Adoptive Transfer of Fluorescent Labeled Lymphocytes
A. Lymphocyte Isolation from donor mice:
- Prepare a Styrofoam board lining with paper towels and sprayed with 70% EtOH to disinfect.
- Place a cell strainer in a 5cm dish and add 5ml RPMI-10.
- Euthanize a donor mouse by C02 followed by cervical dislocation.
- Soak the mouse in 70% EtOH for disinfection
- Pin down the mouse on the Styrofoam board and cut skin open as shown in Figure 1.
- Dissect lymph nodes (LNs) with care into the cell strainer without injuring them.
- Mash the LNs in the cell strainer by the black plunger head of a 15ml syringe.
- Transfer the single cell suspension through the strainer into a 15ml tube by the same syringe.
- Spin down lymphocytes at 1500rpm for 5 minutes.
- Discard the supernatant and resuspend in 10mL of RPMI-10.
- Count the total number of lymphocytes. Purify certain cell population if necessary.
- If the lymphocytes do not have fluorescent genetic marker(s), label lymphocytes with a vital dye(s) (e.g. CFSE or CMTMR) of desired color(s).
B. Labeling of Cells with CFSE, CMTMR or efluor
- Resuspend cells @107/ml in RPMI 0%FCS in a 15ml tube
- Add CFSE @ 2.5uM(stock is 10mK-4000x)
Or add CMTMR @ 20uM (stock is 20mM – 1000x) Vortex to make sure that dye is evenly distributed.
- Incubate 30min @ 37C.
- Add 1-2x volume 100%FCS.
Incubate 2min @ RT.
- Wash with R10 twice.
- Wash with PBS.
- Resuspend for injection.
- Wash lymphocytes once with PBS.
- Resuspend in PBS to the desired concentration (usually 5 to 10x106 cells/mouse).
- For tail vein injection: maximum 250ul per mouse can be used.
- For retro-orbital injection: maximum 100ul per mouse can be used.
- Lymphocytes should be able to home to the LNs within hours. To observe maximum number of lymphocytes, inject lymphocytes into the recipient mice at least 12hr before imaging.
Isolation and Mounting of Lymph Node for Imaging
A. LN dissection:
- Prepare a 6-well dish and add 7ml imaging medium/well.
- Set up Styrofoam board as in section A-1 to dissect the LNs from recipient mice.
- Generally, popiteal LNs (PLNs) are most suitable for imaging due to the small size. Take multiple LNs as backup or specific draining LNs as necessary.
- Keep the dissected LNs in imaging medium at room temperature.
- Place a 5cm dish bottom up on the dissection microscope as a working platform.
- Pipet 200ul imaging medium on top of the dish as the working droplet to keep the LN moist.
- Transfer one LN to the droplet and carefully remove fat and excess tissues from the cortical side of the LN so that it is clear for imaging. (Usually the cortical side is side without visible blood vessels while the medullary side is curved inward with visible vessels.)
- Adjust the LN so that the cortical side is facing up and ready for mounting.
B. LN mounting:
- Cut 22mm square plastic coverslip into nine ~7 mm squares.
- Use a 200ul pipet tip to draw ~5ul Vetbond by capillary suction and place a tiny dot onto the 7mm square.
- Remove excess Vetbond by a clean 200ul pipet tip and then evenly smear Vetbond onto the square. Too much Vetbond will create structures when activated in water.
- Remove excess medium from the LN by placing it on dry areas of the dish several times and quickly place LN medullary side down onto the square before the LN dries out.
- Give a gently tap on top of the LN to secure the binding.
- Immerse the square with the LN in a new 6-well with image medium to activate the excess Vetbond on the square.
- Transfer the mounted LN back to platform to remove any bubble around the LN.
- Place the mounted LN back into the well until ready to image.
Ex-vivo Lymph Node Imaging
A. Set up for perfusion/heating chamber.
- Place imaging media container in water bath, set to 37C.
- Tubing should go from imaging media through peristaltic pump, in line heater and into imaging chamber.
- Additional tubing should go to imaging media from a 5% CO2 tank to bubble in CO2.
- Heating chamber should be assembled and connected to correct cables, including 2 temperature probes, 2 heater connections and vacuum line. Vacuum line should go through a flask to collect media waste. On the heater controller, the inline heater should be set near 40C while the stage should be adjusted in order to keep media at 37C. Monitor 1 shows the stage temperature and Monitor2 shows the media temperature.
- Smear vacuum grease on the imaging platform.
B. Place lymph node, coverslip down onto imaging platform. Press down on the coverslip firmly so that the vacuum grease will hold it into place.
C. Locate Lymph Node.
- Center the objective and lower into the media.
- Search for the surface of the lymph node. To image 2nd harmonic, set laser to 910 nm. It should appear on the violet channel (~405).
- To search for the center/top of the lymph node:
- Move the objective up until you can only see part of the surface at the edge of the field of view.
- Move stage so that surface in focus is now in the center of the field of view.
- Repeat steps a - b until you have the center of the lymph node in your field of view.
- Lymph Node Image : http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/imagesbig/Image5.jpg