Visualizing the dynamics of T cell-dendritic cell interactions in intact lymph nodes by multiphoton confocal microscopy

Abstract

Multiphoton microscopy has provided us the ability to visualize cell behavior and biology in intact organs due to its superiority in deeply into tissues. Because skin draining lymph nodes are readily accessible via minimal surgery, it is possible to characterize the intricate interactions taking place in peripheral lymph nodes intravitally. Here we describe our protocol to visualize antigen specific T cell-dendritic cell interactions in the popliteal lymph node of immunocompetent mice. With this method, behaviors of up to four cells types, such as T cells with different antigen specificities, T cells differentiated into different effector and regulatory lineages and dendritic cells originating from mice that bear mutations in functional genes can be imaged simultaneously.

1. INTRODUCTION

Foreign proteins that enter through skin are cleaved into antigenic peptides and presented by dendritic cells (DCs) [1]. Naïve T cells enter secondary lymphoid organs from blood to assume effector functions based on their ability to recognize these foreign antigenic peptides displayed by DCs [2].The recognition is observed initially as a slow-down, then a complete stop in the T cell mobility preceding the effector functions such as cytokine secretion and killing. It’s very important to understand the biology and dynamics of T cell interactions within the intact lymph nodes and because multiphoton microscopy offers great access to deeper structures buried under the capsule such as T cell zone, it has been increasingly used as the gold standard imaging technique to elucidate the contact characteristics of T cells [3].

Here we demonstrate that T cells and DCs isolated from four different sources that are labeled differently, can be imaged simultaneously by the intravital multiphoton microscopy of popliteal lymph node. We show that mature DCs can be freshly isolated from spleens of reporter mouse strains, loaded ex-vivo with antigenic peptides, injected subcutaneously via footpad injections for delivery into the popliteal lymph nodes. Likewise, naïve T cells that are isolated from spleen can be labeled with primary amine dyes and adoptively transferred into the blood stream via retroorbital sinus injection immediately after administering DCs into the footpad lymphatic vessels. This results in a T cell response initiated at the popliteal lymph node which is optimally detectable by intravital multiphoton microscopy starting at 18–20 h post adoptive transfer [3, 4]. Here, we also provide the information about the important anatomical landmarks to guide minimally invasive surgery and stabilization of the organ for imaging. Lastly, we show that the biology of cell-cell interactions can be analyzed by Huygens and Imaris software which can be utilized to create surface rendering for the T cells and DCs to determine their contact morphology and to quantify the contact duration, area and volume.

2. MATERIALS

1. Curved general purpose Forceps and Scissors

2. Absorbent bench covers

3. 70% Ethanol in spray bottle

4. 1.7 mL, 5 mL, 10 mL, 50 mL sterile conical tubes

5. 60 mm cell culture dish

6. 70 μm cell strainer

7. PBS

8. R10 media: RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL penicillin, 50 μM streptomycin, 1 mM sodium pyruvate 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 μM 2-mercaptoethanol and 10 mM HEPES

9. MACS buffer: PBS supplemented with 0.5% BSA, 2 mM EDTA

10. ACK Lysing Buffer for red blood cell lysis

11. Collagenase: Liberase TM (Sigma-Aldrich, catalog number: 5401119001)

    Prepare Collagenase by adding 10 mL of R10 to 50 mg Liberase TM for a final concentration of 5 mg/mL. Keep solution on ice, stir gently every 5 min for 30 min until dissolved. Aliquot in 1 mL volume and store at −20 °C.

12. DNAse I: Prepare DNAse I stock solution by adding 10 mL of 0.15 M NaCl to DNAse I for a final concentration of 10 mg/mL, aliquot into 500 μL and store at −20 °C. Prepare the digestion mixture for DC isolation by mixing 500 μL DNAse stock solution into 1 mL Liberase solution and adding 1 mL of mixed DNAse/Liberase solution into 11 mL complete RPMI medium (R10).

13. Ultrapure mouse CD11c microbeads (Miltenyi Biotec, catalog number: 130-108-338)

14. Naïve mouse CD4+ T cell isolation kit (Miltenyi Biotec, catalog number: 130-104-453)

15. autoMACS Pro Separator (Miltenyi Biotec, catalog number: 130-092-545)

16. Cell Proliferation Dye eFluor^™ 450, Cell Proliferation Dye eFluor^™ 670 (Thermo Fisher Scientific, catalog numbers are 65-0842-85, 65-0840-85 respectively)

17. 21G, 25G needles, 3–10 mL syringe, 0.3 mL 31G insulin syringe with permanently attached needle

18. Isoflurane for anesthesia

19. O₂ chamber

20. Haemocytometer

21. Trypan blue

22. Anti-CD31 antibody (Alexa Fluor-647 labeled, clone MEC13.3)

23. Betadine (or other iodine-based surgical scrub) and 70 % alcohol prep pads

24. Ophthalmic ointment Artelac Nighttime Gel (Bausch & Lomb)

25. Surgical scissors and dressing forceps

26. Small animal hair clipper MiniArco Professional Cordless Trimmer

27. Stainless steel tissue holder (NIH Division of Scientific Equipment and Instrumentation Services) (see Note 1)

28. Cyanoacrylate (e.g. superglue)

29. Carbomer-based gel (see Note 2) [5]

30. Surgical cloth tape

31. Cotton swab Q-tips

32. Rodent warmer/Heating pad (Braintree Scientific)

33. Isoflurane for anesthesia

34. Isoflorane anesthesia vaporizer (SurgiVet) with mouse nose cone (Braintree Scientific)

35. Leica MZ95 Surgical Microscope

36. Leica SP8 confocal microscope equipped with: two infrared pulsed lasers – a Mai Tai HP laser for 700 – 950 nm excitation and a Spectra Physics Insight DS laser for 950 – 1300 nm excitation; and four channel RLD dector (non-descanned detector) containing two HyD dectors and two photomultipler tube detectors.

37. Huygens Software Suite

38. Imaris Software

3. METHODS

3.1. DC isolation and peptide loading

1. Follow steps 2 – 8 to remove the spleen.

2. Euthanize the mouse and place it face up on an absorbent bench cover and spray abdomen area liberally with 70% Ethanol.

3. Pinch the skin at the midpoint of the abdomen (Fig. 1a) and make a 1 cm incision with curved scissors.

4. Hold both sides of incision and retract the skin in opposite directions (Fig. 1b) (see Note 3).

5. Hold the peritoneum at the left upper quadrant of the abdomen with forceps and make a 5 mm incision and enlarge it to 1–1.5 cm have a better access to spleen.

6. Gently, lift the spleen with forceps to see its ligaments and surrounding connective tissue.

7. Carefully cut the ligaments, remove connective tissue and fat as much as possible.

8. Put the spleen in 60 mm dish that contains R10 media on ice and move to the next spleen.

9. Insert the needle along the long axis of spleen until one third of the axis (Fig. 1c). (see Note 4)

10. Flush spleens one by one with ~2.5 mL digestion mixture/ spleen using a 21G needle and forceps to remove the connective tissue. (see Note 5)

11. Using forceps and curved scissors, cut the spleens into small pieces. (see Note 6)

12. Aspirate the media containing spleen pieces with a 25 mL pipette and put into a sterile 15 mL conical tube. (see Note 7)

13. Incubate for 30 min at 37°C. During the incubation, vortex for 10 seconds at high speed every 5 min

14. Place a 70 μm cell strainer into a new 60 mm dish and mash spleens through it by the help of a sterile syringe plunger. (see Note 8)

15. Pellet the cells by centrifuging at 500 × g for 5 min and discarding the supernatant. (see Note 9)

16. Add 4 mL ACK lysis buffer, resuspend the pellet up and down a few times and put on ice for 3 min. (see Note 10).

17. Add 10 mL MACS buffer to stop the ACK lysis, centrifuge the tube at 500 × g for 5 min and discard the supernatant.

18. Repeat step 17.

19. Resuspend up to 5 spleens in 3 mL MACS buffer.

20. Add 90 μL CD11c microbeads per spleen to label the DCs for magnetic selection, vortex and incubate for 15 min at 4 °C.

21. Top off with MACS buffer to fill the 15 mL tube and centrifuge at 500 × g for 5 min.

22. While pelleting cell/microbead mixture in the previous step, place a 70 μm cell strainer upside-down on top of a new 15 mL tube.

23. After centrifugation, remove supernatant and add 2 mL MACS buffer onto cell pellet, resuspend by gently pipetting up and down and pass the cell-bead mixture through the strainer into the new 15 mL tube. Then rinse the initial tube with 2 mL MACS buffer and apply this through the strainer too (see Note 11).

24. Place the tube that contains cells along with 2 empty tubes labeled as positive and negative into the destined positions on autoMACS rack after removing the caps of all the tubes (see Note 12).

25. Run on autoMACS under the “PosselD2” program.

26. Collect the positive fraction that contains DCs.

27. Pellet DCs by centrifugation at 500 × g for 5 min.

28. Resuspend the DCs in 1–2 mL by gently pipetting up and down and add the peptide to reach the desired loading concentration (see Note 13).

29. Vortex and incubate at 37 °C for 30 min – 3 hrs. (see Note 14).

30. Add 10 mL ice-cold R10 and centrifuge at 500 × g for 5 min, discard the supernatant.

31. Add 10 mL ice-cold PBS and centrifuge at 500 × g for 5 min, discard the supernatant.

32. Resuspend the pellet with 1 mL ice-cold PBS and count the cells.

33. Adjust the volume to reach 50 μL (containing 1–2 × 10⁶ cells) per footpad, gently pipette up and down, transfer into a 1.7 mL tube.

34. Draw the content into 0.3 mL 31G insulin syringe with permanently attached needle. (see Note 15).

Figure 1:

Excision of spleen from euthanized mouse for DC and T cell isolations. Steps for the removal of organ (A-E). Stabilization and flushing of the spleen for DC purification (F).

3.2. Naïve CD4+ T cell isolation and primary amine labeling

1. Remove the spleens as in Fig. 1 and place in 10 mL R10 on ice.

2. Set a 70 μm cell strainer into a 60 mm dish and mash spleens through it by the help of a sterile syringe plunger (see Note 16).

3. Centrifuge at 1500 rpm (500 × g) for 5 min and discard the supernatant (see Note 17).

4. Add 4 mL ACK lysis buffer, resuspend the pellet up and down a few times and put on ice for 3 min. (see Note 18)

5. Add 10 mL MACS buffer and centrifuge the tube at 1500 rpm (500 × g) for 5 min.

6. Resuspend the pellet in 10 mL MACS buffer by gently pipetting up and down, take 10–20 μL to count cell number and centrifuge the rest at 1500 rpm (500 × g) for 5 min.

7. Determine cell number with desired method.

8. Resuspend cell pellet in 40 μL of buffer per 10⁷ total cells and add 10 μL of Biotin-Antibody Cocktail per 10⁷ total cells.

9. Vortex and incubate for 5 minutes in the refrigerator (2−8 °C).

10. Add 20 μL of buffer, 20 μL of Anti-Biotin MicroBeads and 10 μL of CD44 MicroBeads per 10⁷ total cells.

11. Vortex and incubate for 10 minutes in the refrigerator (2−8 °C).

12. Add 10 mL MACS buffer and centrifuge the tube at 1500 rpm (500 × g) for 5 min.

13. Aspirate supernatant completely. Resuspend up to 10⁸ cells in 1 mL of buffer, remove the clumps as in “DC isolation step 23” rinsing the empty tube and the strainer with 1 mL of MACS buffer. Cells are in a final volume of 2–4 mL. Proceed to magnetic cell separation.

14. Place tubes in the following autoMACS Chill Rack positions: position A = sample, position B = negative fraction, position C = positive fraction.

15. Select the program “Depletes” and collect enriched naive CD4+ T cells at position B = negative fraction.

16. Add 10 mL PBS, centrifuge the tube at 1500 rpm (500 × g) for 5 min.

17. Aspirate the supernatant, resuspend in 1–2 mL PBS by gently pipetting up and down and proceed to primary amine labeling (see Note 19).

18. Reconstitute one vial of Cell Proliferation Dye eFluor^™ 670 to a stock concentration of 5 mM with 126 μL of anhydrous DMSO and one vial of Cell Proliferation Dye eFluor^™ 450 to a stock concentration of 10 mM with 165 μL of anhydrous DMSO (see Note 20).

19. Prepare a 20 μM (1/500) solution of Cell Proliferation Dye eFluor^™ 450 or 10 μM (1/500) solution of Cell Proliferation Dye eFluor^™ 670 in PBS pre-warmed to room temperature and cover with foil.

20. Add the 1/500 dye solution at equal volume to the cell suspension to obtain 1/1000 final concentration of the dye.

21. Vortex the cell-dye mixture and incubate for 10 minutes at 37 °C in the dark.

22. Stop labeling by adding 10 mL cold R10 and centrifuge the tube at 1500 rpm (500 × g) for 5 min.

23. Wash cells 2 more times with complete media and for a third time with PBS.

24. Count and adjust the volume accordingly for 4–10 × 10⁶ cells/ 100 μL in PBS (see Note 21).

25. Transfer into a 1.7 mL tube, draw into a 1 mL syringe with 25G needle and place the syringe on ice (see Note 22).

3.3. Adoptive Transfer

1. Anesthetize animals in the anesthesia induction chamber with 2% isofluorane USP (Baxter) admixed with 3 mmHg oxygen. (Note: Watch as their breathing becomes shallow and rapid and wait for the breathing to become deeper and slower to quickly proceed with the injections.)

2. Insert the 31G needle underneath the digital footpad until tip of needle reaches metatarsal footpad as described in Fig. 2a (see Note 23).

3. Inject 50 μL and wait for three seconds before removing the needle. (Note: As you remove the needle, it is normal to see a drop of clear fluid coming out of the injection site. It shouldn’t contain any blood).

4. Position the animal as in Fig. 2b and quickly proceed to retroorbital injection before anesthesia wears off (see Note 24).

5. Glide the needle through inner commissure of eye underneath the eyeball until the needle opening disappears (see Note 25).

6. Inject 100 μL and wait for three seconds before removing the needle (see Note 26).

7. Return animals to their cage until the microscopy (see Note 27).

Figure 2:

Footpad and retroorbital injections for DC and T cell adoptive transfers respectively.

3.4. Minimally invasive surgery for intravital microscopy (IVM) of Popliteal Lymph Node

Intravital imaging of the mouse popliteal lymph node requires minor surgery; however, this makes it an invasive intravital imaging method and therefore is performed as a non-survival technique.

1. Induce animal anesthesia in induction chamber using 2 % isoflurane. Following initial anesthesia, maintain isoflurane influx using a nose cone, at a concentration of ~1.5–1.75 % (see Note 28).

2. Inject the mouse with Alexa Fluor-647 labeled anti-CD31 antibody i.v. via retroorbital route to visualize the blood vessels (see Note 29).

3. Place the mouse on a heated pad under the surgical microscope and secure the mouse in ventral recumbency using surgical tape (Fig. 3A, left) (see Note 30).

4. Remove all hair from mouse leg and thigh using hair clipper.

5. Disinfect the surgical site with 3 alternating scrubs of Betadine (or other iodine-based surgical scrub), and 70% ethanol.

6. Position and focus the binocular microscope on the mouse hind limb. Make 1-cm-long incision on caudomedial side of the knee (Fig. 3A, right), gently pull skin flaps apart exposing semimembranosus muscle and gastrocnemius muscle. Using blunt dissection, separate the muscles to expose the popliteal lymph node. Using micro-dissecting forceps, carefully separate the fat surrounding the lymph node taking care to avoid damage to the arteries, veins, and lymphatic vessels (Fig. 3B).

7. Remove mouse from under the surgical scope, and glue tissue holder to the muscles around exposed lymph node, with the lymph node facing the imaging window (see Note 31).

8. Immediately after attachment of muscle tissue to the holder, immerse the lymph node in carbomer-based gel. Do not allow lymph node surface to dry (Fig. 3C).

9. For the IVM, flip the holder face down onto the microscope imaging stage, adding more gel as needed, and immobilize the entire mouse leg on the imaging stage using surgical tape (Fig. 3D). Cover the edges of the tissue holder with PBS (37°C) -soaked gauze to prevent any drying (see Note 32).

10. Keep the imaged animal inside the 37°C-heated microscope chamber, placing the temperature probe as close to the animal as possible. That will prevent both hypothermia and overheating of the sedated animal. The duration of imaging should vary from 30 minutes to six hours maximum.

11. Once imaging is complete, euthanize the mouse by cervical dislocation while the animal is still anesthetized.

Figure 3:

Animal preparation for surgery. A) The mouse is anesthetized and restrained (left), and 1-cm incision on caudomedial side of the knee is performed (right) to access the popliteal lymph node. B) A drop of superglue is applied to the tissue holder around its middle window, and the muscles surrounding exposed lymph node are attached to the holder, with the lymph node in the center of the window. C) Carbomer-based gel is applied to exposed mouse tissues. D) The mouse is positioned onto the microscope platform, and tissue holder is restrained using surgical tape, with the lymph node facing inverted microscope objective, for time-lapse imaging.

3.5. Acquisition for intravital microscopy (IVM) of Popliteal Lymph Node

1. On the Leica SP8 confocal microscope or equivalent, tune the Mai Tai laser to 880 nm and InSight DS laser to 1150 nm wavelengths for simultaneous excitation of collagen fibers as second harmonic generation (SHG) signal, eGFP, dsRed and e670 dye. (Keep in mind these laser can be tuned to other appropriate wavelengths if using different fluorescent protein/dye combinations such as replacing SHG with an e450 dye).

2. Allow both the mouse and the motorized stage to reach complete temperature equilibrium before acquiring time-lapse video.

3. Define region of interest (ROI) within T cell zone and set a z-stack over time for imaging a single ROI or set a tiled scan of multiple ROIs if imaging larger area (up to 2 mm²). Number of optical slices within a z-stack and the size of imaged area must be optimized to allow appropriate acquisition speed.

4. While using 25x water-immersion lens, the water drop between objective and imaging stage must be refilled every hour. Sequential 1 h time-lapse videos can be combined later into continuous 6 h time-lapse during post-acquisition data processing using Huygens Professional Software.

3.6. Data analysis for intravital microscopy (IVM) of Popliteal Lymph Node

3.6.1. Correct thermal drift and improve image quality by applying deconvolution

1. Correct for image drift due to thermal changes and other shift or rotation errors using the Huygens Professional Software suite by following steps 2 – 4.

2. Stabilize 3D slices in z direction using “Stabilization Wizard” (Fig. 4) (see Note 33).

3. Stitch and stabilize image time series using “Object Stabilizer” cross correlation algorithm of the software to minimize shifts and thermal drift deformations between adjacent time frames (Fig. 5). Images are converted to 32-bit float format. (see Note 34).

4. Deconvolve the image using the “Deconvolution Wizard” (Fig. 6). (see Note 35).

Figure 4:

Huygens Stabilization Wizard showing XY, XZ and YZ projections of the image volume and correlation selection for rotation detection and iterative filtering during one of the wizard steps.

Figure 5:

Object Stabilizer of Huygens software using cross correlation to normalize thermal drift of 4D dataset. The cropping frames for all time points based on correlation correction are shown on XY, XZ and YZ projection as well as total displacement in x direction in voxels. Outliers are adjusted to eliminate one frame with extreme shift.

Figure 6:

Deconvolution wizard of Huygens software. Channels are split and deconvolved separately using the microscopic template and defining deconvolution template. Window on the left shows channels and their deconvolution progress, yellow window in the middle is deconvolution log showing iteration process and information messages. Top right images show original channel image and changes of the same image during the deconvolution. Deconvolved channels are fused together and saved at the end of the deconvolution wizard.

3.6.2. Track cells and calculate colocalization

1. Open the stabilized data in the Imaris software package to proceed with cell tracking, 3-D reconstruction and surface modelling.

2. Select the “Spot” function to create spots of different sizes representing different populations of cells using fluorescent information of each channel. (Fig. 7) (see Notes 36 and 37)

3. In this study we used Imaris plugins called XTensions written in Matlab. Use XTension of Imaris software called “Colocalize Spots” and set the threshold for cell to cell distance as 10 μm. (Fig. 8) (see Note 38).

4. Calculate the spot-spot colocalization parameters such as number and duration of contacts and export them as excel files (Fig. 9).

Figure 7:

A) Image of the 3D reconstructed volume in Imaris shows all cell populations in different channels/colors. Green represent YFP+ DCs; blue, red and magenta represent naïve T cells with different antigen specificities. B) Image shows spots of different sizes created from previous image using Spot function of Imaris and representing different populations of cells.

Figure 8:

Imaris “Spots” function and tracking alghoritm for spots are applied sequentially. Image shows tracks of all cell populations in time. Real channel volume signal of the cells and spot objects are overlaid.

Figure 9:

A) Image shows only colocalized spots representing different spots populations shown in different channel colors and sizes. On the left side XTension “Colocalize Spots” selection is shown as well as groups of colocalized spots created by this algorithm. Images show tracks of colocalized spots between red and magenta cells (B), tracks of colocalized spots between blue and red cells (C). Real channel volume signal of the cells and spot objects are overlaid.

3.6.3. Calculate the colocalization and cell-cell distance

1. Select “Surface” function to reconstruct the 3D structure of the cells using fluorescent signals. (Fig. 10 & 11) (see Note 39).

2. Use XTension “Distance Transformation” to calculate distances of every cell from the surfaces of other cell types represented in different channels at any time point of the time sequence. (Fig. 11) (see Note 40).

3. Select “Surface-Surface Colocalization” XTension to calculate 3D surface volume overlap between interacting cells in time and export the data. (Fig. 12) (see Note 41).

Figure 10:

A) Image of the 3D reconstructed volume in Imaris shows all cell populations in different channels/colors. B) The same image represented as 3D surface models of the cells in Imaris.

Figure 11:

Distance transform channel generated by XTension in Imaris is shown in white color on top of cell volumes in Imaris. Green cell surfaces (on the left image) were used to create distance transform channel outside of the green cells, the minimal distance of the other cells (image on the right) was determined as a minimal intensity of the white distance transform channel between green and other channel surfaces.

Figure 12:

Images showing localization of surface overlap created by “Surface-Surface Colocalization” XTension between red and green cell in Imaris. Image on the top left shows cells in 3D volume, on the top right in reconstructed 3D surface models, on the left bottom with surface overlap shown as a white surface object generated by XTension. Image on the bottom right show the surface overlap after removing green cell surfaces.

4. NOTES

1. We use a custom-built 2 cm × 5 cm stainless-steel plate with a circular window in the middle. After animal preparation, the window will face water-immersion lens of inverted microscope platform. The tissue holder was designed to stabilize small area within larger animal organ by gluing the organ to the stage and allowing the region of interest to be exposed through the opening.

2. We use pre-warmed carbomer-based gel, pH buffered, and matching refractive index of water. This allows exposed animal tissues remain lubricated for hours of imaging and does not interfere optically with using 25x water lens.

3. Slightly turn the mouse on its right side to view the spleen better.

4. Stabilizing the needle between the tongs of forceps helps flushing.

5. After injecting 1.25 mL of the digestion mixture, turn the spleen 180 degrees and inject the next 1.25 mL from the other end of spleen to equally flush all sites. 12 mL digestion mix is enough for up to 5 spleens, so do not combine more than 5 in one tube. 1 spleen yields between 0.5 × 10⁶ – 1.5 × 10⁶ purified DCs depending on the strain and age of the wild-type mice. Spleens from 12 weeks old C57BL/6 mice yield approximately 1.0 × 10⁶ DCs/ spleen.

6. Mince into smallest pieces possible.

7. Make sure to use 25 mL pipette, as the pieces may clog the 10 mL pipette.

8. Apply the pressure using rubber end of plunger. Once all the pieces are mashed, aspirate the cell suspension from the dish using a sterile pipette and pass through the strainer to get rid of clumps.

9. Aspirate the supernatant by vacuum without disturbing the pellet.

10. Keeping cells in ACK buffer longer than 5 min may destroy the cells other than RBCs.

11. This step would remove all the cell clumps that may otherwise clog the autoMACS.

12. Store the reusable autoMACS racks in refrigerator at 4 °C until separation.

13. Different peptides can be combined at this stage if the ultimate aim is to load DCs with multiple antigens.

14. Different peptides have different times for optimal loading. Usually, I-Ab restricted peptides such as OVA_(323–339), LCMV GP_(61–80), Ea_(52–68) etc. would be optimally loaded within 45 min.

15. Insulin syringes with permanently attached needles don’t have dead space; therefore, you only need to draw an extra 50 μL. Remove all the air bubbles by flicking the syringe with fingers and push the bubbles out.

16. Mash up to 3 spleens at once in a cell strainer. Set another petri dish with cell strainer if you have more than three spleens. Once all the spleens are mashed, aspirate the cell suspension from the dish using a sterile pipette and pass through the strainer to get rid of clumps.

17. Aspirate the supernatant by vacuum without disturbing the pellet.

18. Keeping cells in ACK buffer longer than 5 min may destroy the cells other than RBCs.

19. Wrap the tube with aluminium foil as the next steps will contain light sensitive material.

20. Once reconstituted the dye should be protected from light and stored with desiccant at less than or equal to −20°C. Therefore, prepare 5 μL aliquots to avoid freeze-thawing.

21. The cell concentration should be adjusted in accordance with the DC concentration to aim a DC: T cell ratio of 1/4 or 1/5.

22. Take into account the 100 μL dead space, therefore you only need to draw 200 μL extra minimum. Also make sure to remove all the air bubbles by flicking the syringe with fingers and also pushing the bubbles out.

23. Try not to severe the veins that run parallel to the route of footpad injection. If you accidentally hit the vein, do not proceed with the injection as there will be extravasated blood that fills the subcutaneous space.

24. If you notice that animal starts moving and/or breathing rapid and shallow, return it into the anesthesia chamber, and wait until muscle movements disappear before proceeding to the injection.

25. Insert the needle with 30 degrees angle to the bench. You shouldn’t feel any resistance while doing that, if so, you might have inserted the needle too far.

26. As you remove the needle, it is normal to see a drop of blood coming out of the injection site. If any clear fluid pops out as you inject, it means your injection did not hit the vein thus was unsuccessful. Proceed with the other eye.

27. If this preparation starts in the morning on the day before the intravital microscopy, the adoptive transfer can be done by 5.00 pm. This would allow the preparation of animal for microscopy to start at 10.00 −11.00 am the next morning.

28. For intravital imaging, anesthesia can be delivered either as an injectable (avertin or ketamine/zylazine) or inhaled (isoflurane). While each anesthetic has advantages and disadvantages for imaging, isoflurane anesthesia typically yields the most consistent results, and does not require multiple injections or a catheter application for over 30-min time-lapse recording.

29. We typically inject 25 μg per mouse in total volume not to exceed 100 μL.

30. Either upright or inverted microscope setup can be used for multiphoton intravital imaging [6]. While using upright microscope is a well-established technique [4], we use inverted microscope as the one that has number of advantages. Traditionally, inverted microscopes are used for life science research, because gravity makes mouse tissues adhere closely to the coverglass and produce even and stable field of imaging, which is especially crucial during in vivo recording of popliteal lymph node.

31. Extra care should be taken to prevent the adhesive from entering the imaging area.

32. PBS and carbomer gel should be warmed to 37 °C prior to application to the mouse. The addition of cold buffers alters the movement of cells within the tissue.

33. Use “Rotation Detection” if your 3D tiles are slightly rotated due to the thermal drift and iterative filtering parameter if many slices at the edges are out of focus.

34. Please adjust the outliers value if your time series does not contain many outliers and you want to avoid significant data cropping.

35. It is important to acquire images with good sampling rate, as closest to Nyquist rate as possible. Please verify microscopic parameters in deconvolution software before you start deconvolution and make sure everything is correct. This is critical for estimating best PSF for deconvolution. Also make sure you adjust the background subtraction and signal to noise ratio avoiding artefacts.

36. Make sure you select the radius and quality threshold parameter for spots to cover most of the cells in 3D.

37. To determine the dynamic behavior of each cell type described by spots, you can use “Track spots” feature and export statistics such as average track speed, length, straightness etc. as excel files.

38. XTension Colocalize Spots measures the distance between the selected groups of spots and based on the threshold value and divides the spots into two groups: “Colocalized” and “Non-colocalized”. Colocalized spots are located in the same area or very near of each other specified by threshold. Please select the threshold value that best suits the size of the cell populations in your dataset.

39. Each cell population require specific quality threshold and smoothness for surface creation. Make sure you select the threshold that best suits your cells and creates best surface model covering all cell volumes. For splitting the cell surfaces enable “Split touching Objects (Region Growing)” function in connection with specific “Seed Point Diameter”. Eliminate extreme small volumes as debris in the last step of the surface creation wizard.

40. After activating the specific surface “Distance Transformation” XTension, select “Outside of the object”. The XTension automatically converts image into 32-bit float data type (in case it is not 32-bit already) and calculates a mesh of distances from the surface objects starting at the surface and continuing outside of the cell.

41. “Surface-Surface Colocalization” XTension creates masks of two selected surface scenes. It finds the voxels (pixel represented as unit volume in a 3D matrix) inside each surface that overlap with another surface and creates a new channel. Software then uses it to create new surface generated from overlapping regions. Colocalized surface should be generated without smoothing factor.