Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

The procedures described herein were approved by the Institutional Review Board of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 2.751.345). Blood samples were obtained from healthy volunteer donors. Animal experimental procedures were conducted in accordance with the Ethical Principles in Animal Research adopted by the Brazilian law 11.784/2008 and were approved and licensed by the Ethical Committee for Animal Research of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 014/2019).

1. Isolation and differentiation of human dendritic cells

1. Pipette 10 mL of the density gradient medium in 50 mL centrifuge tubes.
2. Label the 50 mL tubes according to each donor's sample.
3. Collect ~ 50 mL of venous blood from healthy donors and after collection, follow the procedures described below in the flow cabinet.
4. Carefully transfer the collected blood into centrifuge tubes and dilute in saline solution (0.9% sodium chloride) in a ratio of 1:1 at room temperature.
5. Slowly overlay the diluted blood on the top of the density gradient medium.
6. Centrifuge tubes containing the blood and density gradient medium at 400 x g for 30 min at room temperature.
   NOTE: Switch off the brake before centrifuging to prevent the mixing of gradient layers. After the 1st centrifugation, lower the centrifuge temperature to 4 °C.
7. Carefully remove the tubes from the centrifuge.
8. Identify the ring of peripheral blood mononuclear cells (PBMC) in the sample (buffy coat); gently remove the residual plasma with a pipette.
   NOTE: After centrifugation, the following gradient layers will have formed: erythrocytes, density gradient medium, PBMC ring, and plasma. The PBMC ring is located between the density gradient medium and plasma layers.
9. Transfer the cloud-like PBMC layer to a new tube and bring up the volume to 30 mL with saline solution.
10. Centrifuge the tubes containing cell suspension at 250 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 1 mL of saline solution.
11. Collect an aliquot for cell counting with the trypan blue exclusion method. First, dilute at a ratio of 1:1,000. Then, use 10 µL of the diluted cells for trypan blue staining and count cells using a Neubauer chamber to determine cell viability.
12. Centrifuge again at 200 x g for 10 min under 4 °C.
13. Resuspend the pellet in MACS buffer. Use 80 µL of the buffer for every 1 x 10⁷ cells.
    NOTE: MACS buffer composition: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA. Dilute the buffer solution at a 1:20 ratio with the rinsing solution. Keep the buffer cold and store at 2-8 °C.
14. Add CD14 microbeads to the cell suspension prepared in step 1.13. Use 20 µL of CD14 microbeads for every 1 x 10⁷ cells.
    NOTE: CD14 microbeads are used for the positive selection of human monocytes from PBMCs, as beads bind to CD14-positive cells expressed on most monocytes.
15. Homogenize the solution containing the pellet and microbeads using a pipette. Keep on ice for 15 min.
16. Centrifuge the suspension at 300 x g for 10 min under 4 °C.
17. Resuspend cells in MACS buffer. Use 1-2 mL for 1 x 10⁷ cells in the cell microbead mixture.
18. Centrifuge the suspension at 300 x g for 10 min under 4 °C.
19. Aspirate the supernatant and resuspend the pellet in 500 µL of MACS buffer.
    NOTE: This is the maximum volume of the cell suspension that can be processed through one column.
20. Assemble the magnetic column.
21. Wash the column once by adding 500 µL of MACS buffer. Allow buffer to flow under gravity through the column.
    NOTE: Do not allow the column to dry, as any air that enters can obstruct the column.
22. Add 500 µL of the cell sample solution from step 1.19 per column. Allow the cell sample solution to flow under gravity through the column.
23. Wash the column by adding 500 µL of MACS buffer (2x). Only add the fresh buffer when the column reservoir is empty. Avoid drying.
24. Pipette 1 mL of the MACS bufferonto the column and place it in a new tube underneath. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
25. Centrifuge the CD14 enriched cells at 300 x g for 10 min under 4 °C.
26. Count cells using a Neubauer chamber.
27. Resuspend cells in 1 mL complete RPMI with interleukin-4 (IL-4) (100 µL/mL) + granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL).
    NOTE: GM-CSF is a cytokine secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts that induces the differentiation and proliferation of myeloid progenitors in the bone marrow. GM-CSF and IL-4 are used to induce dendritic cell differentiation³².
28. Seed cells in a 24 well plate at a concentration of 2 × 10⁵ cells per well in 500 μL of complete RPMI medium containing and incubate the cells for 7 days in the cell culture incubator at 37 °C.

2. Leishmania infantum  cultivation

NOTE: Leishmania infantum (MCAN/BR/89/BA262) parasites are used in this assay. Hamsters were intravenously infected with 20 µL of the solution containing 1 x 10⁶ L. infantum promastigotes in sterile saline. After 1 to 2 months, animals were euthanized and the amastigote forms of Leishmania were recovered from their spleens and differentiated into promastigotes³³.

1. Grow L. infantum promastigotes isolated from previously infected hamster spleens in an inclined 25 cm² cell culture flask containing 3 mL of Novy-Nicolle-MacNeal medium (NNN) and 5 mL of supplemented Minimum Essential Medium (MEM).
   NOTE: In this assay, MEM medium was supplemented with 10% fetal bovine serum (FBS) and 24.5 nM hemin bovine.
2. Incubate at 24 °C for 7 days in BOD (Bio-Oxygen Demand) incubator.
3. Pipette 100 µL of promastigote culture into a new 25 cm² cell culture flask containing 5 mL of supplemented MEM medium.
4. Incubate the promastigote culture at 24 °C in BOD incubator until promastigotes reach the stationary phase. Periodically count cultured promastigotes by transferring an aliquot of parasite suspension diluted in saline to a Neubauer chamber (i.e., hemocytometer) to determine the stationary phase of growth.
   NOTE: It is not recommended to use first-passage parasites after cultivation in NNN medium to avoid residual traces of rabbit blood.
5. Transfer 1 x 10⁵ of stationary growth phase culture of L. infantum promastigotes to a new 25 cm² cell culture flask and add 5 mL of MEM supplemented medium.
6. Monitor the growth of the culture periodically for approximately 7 days using a Neubauer chamber until the stationary phase is achieved. Parasites are now ready for use in experimental infection procedures.
   ​NOTE: Promastigote cultures are considered viable for infection for up to 7 passages in vitro; additional passages may induce loss of virulence.

3. Human dendritic cell infection

1. Inside a biological laminar flow cabinet, transfer all the contents from the parasite culture flask to 50 mL centrifuge tubes.
2. Add cold saline solution to a final volume of 40 mL.
3. Centrifuge at 1,600 xg for 10 min under 4 °C.
4. Discard the supernatant following centrifugation and resuspend the pellet in 1 mL of cold saline solution (Repeat steps 3.3-3.4 three times).
5. After washing the cells to remove any non-viable parasites, resuspend the pellet in 1 mL of cold saline solution.
6. Pass the contents slowly through a 1 mL syringe with a 16 G needle 5 times to separate the parasite clusters.
7. Remove an aliquot to determine the parasite concentration in a hemocytometer (calculate the mean number of parasites in 4 quadrants x dilution factor x 10⁴).
8. Wash dendritic cells by adding 1 mL of saline solution and centrifuging cells at 300 x g for 10 min at room temperature (3 times).
9. Calculate the quantity of L. infantum required for the experimental infection; ratio: 20 parasites per cell.
10. Place the required volume of L. infantum in each well of cell culture plates.
11. Incubate dendritic cells with parasites for 4 h in an incubator at 37 °C under 5% CO₂.
12. After step 3.11, centrifuge plates at 300 x g for 10 min under 4 °C.

4. Migration assay using cell culture inserts

1. After infection, wash dendritic cells (infected or not) 3 times with 1 mL of saline solution at room temperature to remove non-internalized parasites. After each wash, centrifuge plates at 300 x g for 10 min under 4 °C.
2. Once non-internalized parasites are removed, detach cells by adding 200 µL of the cell dissociation reagent in each well and incubate for 15 min. Keep the cells in an incubator at 37 °C under 5% CO₂.
3. Homogenize cells using a 1,000 µL pipette tip to allow cells to loosen.
4. Transfer the dissociated cells to a 50 mL centrifuge tube.
5. Centrifuge at 300 x g for 10 min under 4 °C.
6. Resuspend the pellet in 1 mL of RPMI medium supplemented with 10% FBS.
7. Pass the contents of each tube slowly through a 1 mL syringe with a 25 G needle 5 times to separate cells.
8. Remove an aliquot, dilute using trypan blue to determine the cell concentration using a hemocytometer (mean of viable cells in 4 quadrants × dilution factor × 10⁴).
9. Preparation of cell culture microplate inserts:
   NOTE: Cell culture inserts (5.0 µM pore polycarbonate membrane) are recommended for migration assays using dendritic cells, macrophages, and monocytes, as these inserts allow phagocytes to pass through the membranes.
   1. Remove inserts with sterile tweezers and place in empty wells.
   2. Add 600 µL of RPMI medium supplemented with 10% FBS to each well; add chemoattractant chemokine (C-C motif) ligand 3 (CCL3) (1 µL for every 1 mL of medium).
      NOTE: CCL3 is a chemokine that regulates DC migration³⁴.
   3. Using sterile tweezers, place inserts in wells containing the medium.
10. Add 2 x 10⁵ dendritic cells (infected or not) in 100 µL of medium to each insert.
11. Incubate for 4 h to allow cells to migrate.
12. After 4 h, take the plate out and aspirate the medium. Add 100 µL of 4% paraformaldehyde and incubate for 15 min.
13. Remove paraformaldehyde and add 100 µL of saline solution.
    NOTE: Plates can be kept at 4 °C for later assembly.
14. Collect the supernatant from inserts or from wells to count non-migrating cells or those that crossed the membrane, respectively. Incubate with paraformaldehyde 4% for 15 min.
15. Concentrate cells using a cytocentrifugation technique³⁵.
16. Add 10 µL of the mounting medium with DAPI to the membranes and place coverslips over the wells.
17. Assembly of the insert membrane.
    NOTE: This step can be performed outside the biosafety cabinets.
    1. Remove insert membranes from wells.
    2. Scrape the surface of the insert with a swab to remove any cells that have not migrated.
    3. Using a scalpel, remove the membrane from the insert.
    4. Place the membrane on a slide, with cells facing up.
    5. Add 10 µL of mounting medium with DAPI to each membrane and then place overlay coverslips.
    6. Cover plates with aluminum foil.
    7. Incubate plates at room temperature for 30 min, then store at -20 °C
18. To analyze cell migration, count the number of cells per field (no less than 10 fields) using a fluorescence microscope at a laser excitation wavelength of 405 nm.

5. Adhesion assay and evaluation of actin polymerization by immunofluorescence

NOTE: For this assay, use 24-well plates with coverslips.

1. After infecting cells for 4 h, wash each well 3 times with sterile saline solution at room temperature to remove any non-internalized parasites.
2. Centrifuge the plate at 300 x g for 10 min under 4°C. Add 500 µL of 4% paraformaldehyde to each well for 15 min.
3. Remove paraformaldehyde and add 1 mL of saline solution.
4. Prepare solutions as described in Table 1.

Table 1: Buffer recipes.

5. Immunostaining
   ​NOTE: The following steps must be performed under agitation.
   1. Wash coverslips 3 times with 1x PBS for 5 min.
   2. Add 500 µL of ammonium chloride solution per well for 10 min.
   3. Wash coverslips 3 times with 1x PBS for 5 min.
   4. Permeabilize the membrane with Saponin 0.15% for 15 min.
   5. Incubate cells with the blocking solution (PBS 1x / ABS 3% / Saponin 0.15%) for 1 h.
   6. Wash coverslips 3 times with PBS 1x/Saponin 0.15% for 5 min.
   7. Dilute primary antibodies in PBS 1x /ABS 1% /Saponin 0.15% as: Rabbit anti-FAK (pTyr397): 1:500 dilution, Rabbit anti-paxilin (pTyr118): 1:100 dilution. Mouse Anti-Rac1: 1:100 dilution. Rabbit Anti-Cdc42: 1:200 dilution. Rabbit Anti-RhoA: 1:200 dilution.
   8. Incubate cells with primary antibody diluted in PBS 1x/ABS 1% /Saponin 0.15% for 1 h.
      NOTE: FAK and Paxillin are key molecules involved in the formation of adhesion complexes^13,14,15. The Rho GTPase family is responsible for the organization of the actin cytoskeleton. RAC1 and Cdc42, located at the front edge of the cell, control the formation of lamellipodia and filopodia, respectively; RhoA is located at the rear of the cell and is involved in cytoskeleton contraction^{15, 23,24,25}.
   9. Wash coverslips 3 times with 1x PBS/Saponin 0.15% for 5 min.
   10. Dilute secondary antibody or phalloidin as follows: Anti-rabbit IgG, Alexa Fluor 568: 1:500 dilution; Anti-mouse IgG, Alexa Fluor 488: 1:500 dilution; Anti-mouse IgG, Alexa Fluor 594: 1:500 dilution; Phalloidin Alexa Fluor 488: 1:1200 dilution.
   11. Incubate cells with secondary antibody or phalloidin diluted in 1x PBS/ABS 0.3% Saponin 0.15% for 45 min.
       NOTE: Incubation of the secondary antibody or phalloidin should be performed in the absence of light. Cover plates with aluminum foil to protect from light during the following steps.
   12. Wash coverslips 3 times with 1x PBS for 5 min.
   13. Remove coverslips from wells.
   14. In the absence of light, add 10 µL of mounting medium with DAPI onto clean microscopic glass slides.
   15. Place coverslips with cells facing down to allow contact between cells and DAPI solution.
       NOTE: Cover slides with aluminum foil to protect from light.
   16. Incubate for 30 min at room temperature.
   17. Store at -20 °C.

6. Confocal microscopy, image acquisition, and quantification using FIJI

NOTE: To acquire/capture immunofluorescence images, use a confocal laser scanning microscope. Oil-immersion 63x objective lens is recommended for optimal resolution.

1. Allow coverslips to reach room temperature, protected from light, for at least 30 min prior to image acquisition.
2. Clean the coverslips with absorbent tissue.
3. Add a drop of immersion oil to the objective and place each slide under the microscope.
4. Move the objective until the oil touches the slide.
5. Observe and adjust the microscope focus; select the 63x objective with oil.
6. Turn on lasers at 488 nm, 552 nm, and 405 nm wavelengths.
7. Select image resolution: 1024 x 1024.
8. Click on the Live button, set the Z stack, and press Begin. Repeat the process and then press End.
9. After selecting the Maximum Projection option in the Tool menu, wait for the image to be acquired.
10. Save the experiment.
11. Export images in .lif format on a computer. Use FIJI open-source image analysis software to analyze images.
12. Select the images to be analyzed and select Duplicate Image.
13. To have the image in grayscale, select Image | Adjust | Color Threshold. Select 0 and 255 (saturation).
14. Select the threshold method: Default.
15. Select threshold color: B and W (black and white).
16. Do not select Dark background.
17. Select: Process | Binary | Options and then select the relevant data to be measured: Area, Min, Max, gray value, integrate density.
18. Select the free hands' tool in the Fiji toolbar and trace each cell manually carefully.
19. From the Analyze menu, select Set measurements. Make sure to have area integrated intensity and mean grey value selected. Repeat this process for each cell.
20. Select all data in the Results window and copy and paste into a spreadsheet file.
21. Calculate the corrected total cell fluorescence (CTCF): CTCF (Integrate density = Area of selected cell x Mean fluorescence of background readings).
22. Open the file containing data using statistical analysis software to perform statistical analysis.

7. Statistical analysis

1. Open a New Project when a welcome dialog appears.
2. Choose the type of graph and medium with SD.
3. Apply the values obtained from the experimental results to the table.
4. Select Descriptive Statistics and choose the option column Statistics [all tests] to analyze data distribution.
5. If data follow a Gaussian distribution, choose the t-test to analyze samples by comparing two pairs. If the distribution is non-Gaussian, analyze data using the Mann-Whitney test.
6. Choose the best graph option for optimal data representation.