In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

NOTE: Cells were obtained from healthy donor volunteers. The procedure described herein was approved by the National Ethics Committee (number 2.751.345)-Fiocruz, Bahia, Brazil).

1. Differentiation of human dendritic cells

1. Pipette 10 mL of polysucrose-sodium triazoate mixture in 50 mL conical tubes.
2. Label the 50 mL conical tubes respectively for each donor.
3. Collect 30 mL of blood from healthy donors and perform all subsequent steps in the laminar flow hood.
4. Carefully transfer the blood into the conical tubes and dilute the blood in saline solution (0.9% sodium chloride) at a ratio of 1:1 at room temperature.
5. Overlay the slowly diluted blood onto the polysucrose-sodium triazoate mixture in the tubes (step 1.1). Centrifuge the tubes once at 400 × g for 30 min at 25 °C.
   NOTE: Turn off the brake before centrifugation to prevent the mixing of gradient layers. After the first centrifugation, decrease the temperature to 4 °C in the centrifuge.
6. Carefully take the tubes out from the centrifuge.
7. Look for the ring formed by peripheral blood mononuclear cells (PBMCs) in the sample (buffy coat); aspirate the residual plasma carefully with a pipette.
   NOTE: Centrifugation leads to the formation of the following gradient layers: erythrocytes, density gradient medium, PBMC ring, and plasma. The PBMC ring is between the density gradient medium and the plasma layers.
8. Transfer the cloudy PBMC layer to another tube and add saline to a final volume of 30 mL.
9. Centrifuge the tubes containing the cell suspensions at 250 × g for 10 min at 4 °C. Discard the supernatant and add 1 mL of saline to resuspend the pellet.
10. Collect an aliquot for cell counting and dilute it 1:1,000. Use 10 µL of the diluted cells for counting the cells by trypan blue exclusion method using a Neubauer chamber to determine cell viability.
11. Spin the suspension again at 200 × g for 10 min under 4 °C.
12. Resuspend the pellet in magnetic-activated cell sorting (MACS) buffer. Use 80 µL of the buffer per 1 × 10⁷ cells.
    NOTE: See Table 1 for the MACS buffer composition. Keep the buffer cold and store it at 2-8 °C.
13. Add CD14 microbeads to the cell suspension prepared in step 1.9. Use 20 µL of CD14 microbeads per 1 × 10⁷ cells.
    NOTE: CD14 microbeads are used to positively select human monocytes from PBMCs, as beads containing human anti-CD14 bind to CD14⁺ cells expressed on the surfaces of most monocytes.
14. Pipette up and down to resuspend the pellets and microbeads uniformly. Keep on ice for 15 min.
15. Centrifuge the suspension at 300 × g for 10 min at 4 °C.
16. Resuspend the cells in MACS buffer. Use 1-2 mL per 1 × 10⁷ cells in the cell-microbead mixture.
17. Centrifuge the suspension at 300 × g for 10 min at 4 °C.
18. Remove the supernatant and resuspend the pellet in 500 µL of MACS buffer.
    NOTE: This is the maximum volume of the cell suspension to be processed in one column.
19. Assemble the magnetic column.
20. Wash the column once with 500 µL of MACS buffer and allow the buffer to flow under gravity through the column.
    NOTE: Take care not to allow the column to become dry, as air can obstruct the column.
21. Add 500 µL of the cell-bead suspension (step 1.19) per column. Allow the cell suspension to flow under gravity through the column.
22. Wash the column with 500 µL of MACS buffer (2x). Add fresh buffer only when the column reservoir is empty. Do not let the column dry.
23. Place a new tube underneath the column, pipette 1 mL of the MACS buffer onto the column, and immediately flush the magnetically labeled cells out of the column by firmly pushing the plunger.
24. Centrifuge the CD14 enriched cells at 300 × g for 10 min at 4 °C.
25. Count the cells using a Neubauer chamber.
26. Resuspend the cells in 1 mL of complete RPMI with 100 µL/mL of interleukin-4 (IL-4) ( (50 ng/mL) + granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL).
27. Seed cells on a 24-well plate at a concentration of 2 × 10⁵ cells per well in 500 µL of complete RPMI medium containing the above cytokines and incubate for 7 days at 37 °C.

2. Leishmania culture

NOTE: L. amazonensis (MHOM/BR88/Ba-125) parasites were used in this assay.

1. Maintain promastigotes on Novy-Nicolle-MacNeal (NNN) blood agar medium with 5 mL of Schneider's complete medium or Minimum Essential Medium (MEM) in a 25 cm² culture bottle in a Bio-Oxygen Demand (BOD) incubator at 24 °C.
   NOTE: To cultivate the promastigote form of the L. amazonensis species, 5 mL of Schneider's complete medium (Schneider ́s Insect Medium containing 10% inactivated fetal bovine serum (FBS) and gentamicin at a concentration of 50 µg/mL) was used.
2. Incubate for 7 days at 24 °C in the BOD incubator.
3. Pipette 100 µL of promastigote culture into a new 25 cm² cell culture flask.
4. Add 5 mL of the supplemented MEM.
5. Incubate at 24 °C in the BOD incubator and periodically count the promastigotes by transferring an aliquot of saline-diluted parasite cell suspension into a Neubauer chamber (i.e., hemocytometer) until stationary phase has been reached.
   NOTE: Do not use the first passage post-NNN Medium for experiments.
6. Transfer 1 × 10⁵ of Leishmania stationary growth phase promastigotes into a new 25 cm² cell culture flask and add 5 mL of the supplemented MEM.
7. Periodically monitor the growth of cultures using a Neubauer chamber until the stationary phase is achieved.
   NOTE: Use promastigote cultures for up to 7 passages in vitro to avoid loss of virulence.

3. Leishmania infection

1. After verifying stationary-phase growth of cultured parasites, remove all contents from culture bottles and place them in 50 mL conical tubes. Add cold saline solution to achieve a final volume of 30 mL.
2. Centrifuge for 10 min at 4 °C at 1,600 × g three times. Discard the supernatant following centrifugation and resuspend the pellet in a cold saline solution.
3. Wash the cells to remove any non-viable parasites, and resuspend the pellet in 1 mL of cold saline. Pass the suspension 5 times slowly through a 1 mL syringe fitted with a 16 G needle to separate parasite clusters.
4. Remove an aliquot from the sample to count parasite concentration in a hemocytometer using Eq (1).
   Parasite concentration = mean of parasites in 4 quadrants × dilution factor × 10⁴ (1)
5. Calculate the amount of Leishmania sp. required for the number of host cells to be plated to maintain a 10:1 parasite: host cell ratio (step 1.27).
6. Wash the DCs by adding 1 mL of saline solution and centrifuging the cells three times at 300 × g for 10 min at room temperature.
7. Place the required volume of Leishmania in each well of the 24-well cell culture plate (see step 3.5).
8. Incubate the DCs with parasites for 4 h in an incubator at 37 °C in a 5% CO₂ atmosphere.

4. Immunostaining for flow cytometry analysis

1. After infection, wash cells twice with 1 mL of saline solution to remove any non-internalized parasites.
   NOTE: The panel with antibody clones and fluorochromes is listed in the Table of Materials.
2. Dilute antibodies (1:50) in fluorescence-activated cell sorting (FACS) buffer (1x PBS with 1% BSA) at a final volume of 50 µL per experimental condition.
   NOTE: It is important to titrate antibodies before experimentation to ensure optimal staining concentrations. In this protocol, 1:50 was used after titration experiments.
3. Prepare a master mix (1x PBS with 1% BSA + antibody mix). Vortex and pipette 50 µL of the mix in wells containing cells.
4. Briefly incubate the DCs with the antibody mix (anti-human CD1a, anti-human CCR7, anti-human CD83, anti-CD11c, anti-human CD209, anti-human HLA-DR) on ice for 30 min, protected from light.
5. Wash cells twice with 1 mL of cold staining buffer (1% FBS in 1x PBS) and centrifuge at 300 × g for 5 min at 4 °C.
6. Aspirate the supernatant and resuspend the pellet in 200 µL of cold staining buffer.
7. Proceed with data acquisition on a flow cytometer.
   NOTE: To ensure optimal conditions for FACS analysis, data acquisition should be performed within 48 h after sample staining. Data were analyzed using FlowJo software.
   1. Open the FlowJo software program and create a new workspace. Add the flow cytometry files to be analyzed by dragging them into the workspace window.
   2. Click on the tube name to select side scatter (SSC) and forward scatter (FSC) parameters.
      NOTE: Flow cytometric analysis of DC maturation was based on the expression of CD1a, CD11c, CD80, CCR7, CD209, and HLA-DR.
   3. Using the polygon gating tool, draw a gate around the DCs. Double-click on the selected gated cells to display a new window. Perform doublet cleanup by selecting the FSC-A and FSC-H parameters. Using the polygon tool, draw a new plot around individual cells.
   4. Double-click on the gated cells and select SSC and CD11c parameters to generate a gate containing CD11c⁺ cells.
   5. Export the cleaned CD11c⁺ cell gate by selecting it. Right-click on the gate and select the Select equivalent nodes option. Right-click and export the gate along with all compensated parameters by saving it to a recently created folder.
   6. Create a new workspace by opening the recently saved files. Click on Workspace and select plugins | Downsample | 30,000 to 50,000 events; press OK.
      NOTE: As TSNE is both a time-consuming and computationally demanding process, this step is recommended to reduce the total number of analyzed events.
   7. Right-click on a downsampled file and select equivalent nodes.
   8. Right-click on a downsampled file and select Export/concatenate.
   9. Click on the concatenate window and select all compensated parameters. Select the concatenated files and click on workspace | plugin | tSNE.
   10. Select the desired parameters (CD1a, HLA-DR, CCR7, CD80, CD209, and CD11c) to create clusters. Press OK.
   11. Click on the concatenated file to visualize the tSNE1 and tSNE2 parameters.

5. Actin immunostaining

1. Plate infected DCs on coverslips placed in 24-well plates. Centrifuge the plates at 300 × g at 4 °C for 10 min. Wash cells 3x with sterile saline after infection at room temperature to remove any extracellular parasites.
2. Incubate the DCs with 500 µL of 4% paraformaldehyde for 15 min at room temperature. Remove the paraformaldehyde and add 1 mL of saline (see Table 1).

6. Immunolabeling

NOTE: Perform the following steps under agitation.

1. Wash the coverslips with 1x PBS for 5 min; repeat 3x. Add 500 µL of ammonium chloride solution per well; incubate for 10 min at room temperature.
2. Wash the coverslips with 1x PBS for 5 min; repeat 3x.
3. Permeabilize the membrane with 0.15% Saponin for 15 min.
4. Block for 1 h with 1x PBS 1x/3% BSA/0.15% saponin. Wash the coverslips with 1x PBS/0.15% saponin for 5 min; repeat 3x.
5. Incubate the cells for 1 h with phalloidin (1:1,200] diluted in 1x PBS/1% BSA/0.15% saponin. Wash the coverslips with 1x PBS/0.15% saponin for 5 min; repeat 3x.
6. Place the coverslips with cells facing down on mounting medium with 4',6-diamidino-2-phenylindole (DAPI). Cover the slides with aluminum foil to protect them from light.
7. Acquire images using a confocal fluorescence microscope with a 63x/1.4 objective.

7. Confocal microscopy acquisition and Fiji quantification

1. Capture immunofluorescence images using a confocal laser scanning microscope (see the Table of Materials).
   NOTE: For best resolution, use a 63x objective oil immersion lens.
2. Protect the coverslips from light and leave them at room temperature for at least 30 min prior to acquisition.
3. Use absorbent tissue to clean the coverslips. Add a drop of immersion oil to the objective and place the slide on the microscope stage. Select the 63x objective with oil, raise the platform until the oil touches the slide, and adjust the focus on the microscope.
4. Open the program and activate the laser wavelengths of 488, 552, and 405 nm.
5. Set the image resolution to 1024 x 1024 pixels.
6. Click on live bottom, set the Z stack, and press Begin. Repeat this process and then press the End button.
7. Wait for the image acquisition to complete, and then select the option Maximum Projection in tools.
8. Save the experiment, and export the .lif format images to a computer.
9. Open the FIJI program, open the experiment in FIJI, and set the view stack to Hyperstack.
10. Select open image files individually | stitch tiles.
11. Select the free hand tool in the Fiji toolbar and trace each cell carefully by hand. Press analyze | measure to visualize fluorescence intensity. Repeat this process for each cell type per group.
12. Save the measurements and export them to a spreadsheet. Open the file containing data using statistical analysis software (see the Table of Materials) to perform statistical analysis.

8. Statistical analysis

1. Open a new project in the software.
2. For data with normal distribution, use Student's t-test; for nonparametric data, use the Mann-Whitney U test.
3. Paste the values obtained from the experimental results in the table.
4. Select descriptive statistics and choose the option column statistics [all tests] to analyze data distribution.
   NOTE: If the data follow Gaussian distribution, choose t-test to examine samples by comparing two pairs. If the distribution is non-Gaussian, analyze data using the Mann-Whitney U test with central tendency measures (means or medians) and measures of variation.
5. Choose the best graph option for optimal data representation.