Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

NOTE: All medium preparation and cell culture techniques are carried out under aseptic conditions using a biosafety cabinet with laminar flow. All culture incubation steps described are carried out using an incubator designed to maintain an atmosphere of 37 °C, 5% CO₂, and 95% humidity.

1. Cell culture

1. Preparation of growth medium
   1. For MSC and LADMAC, add 50 mL of FBS and 5 mL of 100x antibiotic/antimycotic mix to 500 mL of high glucose DMEM.
   2. For the MΦ, add 50 mL of FBS, 100 mL of LADMAC condition medium (prepared following the steps of section 1.2), and 5 mL of 100x antibiotic mix to 500 mL of high glucose DMEM.
      ​NOTE: LADMAC cells produce colony-stimulating factor (CSF-1) necessary to support the growth of the MΦ cells.
2. Preparation of LADMAC conditioned medium
   1. To seed LADMAC cells from frozen stock, add 19 mL of growth medium from section 1.1 in each of the five T75 cm² cell-culture treated flasks and place in the cell culture incubator for 15 min to equilibrate to 37 °C.
   2. Meanwhile, quick thaw one aliquot of 10⁶ frozen cells by incubating in a 37 °C water bath for 2-3 min or until it gets thawed. Add the cells to 9 mL of fresh MSC growth medium in a 15 mL or 50 mL sterile conical tube and centrifuge at 125 x g in a swinging bucket rotor for 5 min.
   3. Aspirate or decant the supernatant, add 5 mL of fresh growth medium, and gently pipette up and down to resuspend the cell pellet.
   4. Add 1 mL of the cell suspension to each of the five T75 cm² flasks using a sterile serological pipette and place them in the cell culture incubator.
   5. Every 2-3 days, add an additional 10 mL of medium over a 7-10-day period. Then, collect the cells and the supernatant into sterile 50 mL conical tubes using a sterile serological pipette and centrifuge at 125 x g for 5 min.
   6. Separate the supernatant from the cell pellet and sterile filter the supernatant using a 0.2 µm sterile single-use vacuum filter unit.
      1. Remove the aspirator assembly from the package and connect using vacuum tubing to the aspirator pump. Pour the supernatant into the top compartment and replace the cover. Turn on the aspirator pump to filter into the bottom compartment.
      2. Prepare aliquots by pipetting into 50 mL sterile conical tubes and store at -20 °C.
   7. To freeze the cell pellet, resuspend in freezing medium at 10⁶ cell/mL, place in a freezing container, and then place them in a -80 °C freezer. After 24 h, transfer to LN₂ storage.
3. Propagate cells in preparation for co-culture
   1. To seed MSC and MΦ cells from the frozen stock, equilibrate 9 mL of appropriate growth medium prepared in section 1.1 in each of the four 100 mm cell culture treated dishes for each cell type and place in the cell culture incubator for 15 min.
   2. Meanwhile, quick thaw the aliquots of 10⁶ frozen cells by incubating them in a 37 °C water bath for 2-3 min or until just thawed. Then, add the thawed MSC cells to 9 mL of fresh MSC growth medium and add the thawed MΦ cells to 9 mL fresh MΦ growth medium in 15 or 50 mL sterile conical tubes and centrifuge at 125 x g in swinging bucket rotor for 5 min.
   3. Aspirate or decant the supernatant and resuspend each pellet in 4 mL of the appropriate fresh growth medium by gently pipetting up and down. Add 1 mL of the cell suspension to each of the four 100 mm dishes for each cell type that have been equilibrated in step 1.3.1.
   4. Return the dishes with cells to the incubator. Change the medium every 2-3 days until 70%-80% confluent.

2. Seed MSC in experimental dishes, Day 1

NOTE: Prior to seeding the MSC, design the experimental 96-well plate layout for fluorescent spectrophotometry and the chamber slide for dynamic imaging. For the 96-well plate, flank experimental wells with wells containing cells but do not use them in the assay. Mark at least four wells to be used for the reagent blank (RBL). See Table 1 for an example 96-well template and Table 2 for an example of a 4-well chamber slide template. Black or white 96-well plates with clear bottoms are recommended. A 1.5 mm borosilicate glass chamber slide is optimal, but the number of chambers can be adjusted dependent on the study design. A summary flow chart of the methods that follow is included in Figure 2.

1. At 70%-80% confluency, aspirate the growth medium from the cultures in 100 mm dishes and add 5 mL of PBS to isolate the MSC.
2. Aspirate the PBS, add 2 mL of 0.05% trypsin/EDTA, and place it in the culture incubator for 3-5 min.
3. After 3 min, check the cell detachment using an inverted microscope. If the cells are detached, continue to the next step; if not, continue incubation for another 1-2 min. Do not incubate longer than 5-6 min.
4. Add 5 mL of fresh growth medium to the dish with the detached cells. Rinse the dish using the trypsin/medium mixture and collect cells into a clean, sterile 50 mL conical tube using a sterile serological pipette.
5. Centrifuge for 5 min at 125 x g. Aspirate the supernatant and resuspend the cell pellet in 10 mL of fresh growth medium by gently pipetting up and down.
6. To count the cells, add 10 µL of the cell suspension to 30 µL of 0.4% trypan blue solution in a 1.5 mL microcentrifuge tube. Then, pipette 10 µL of the mixture under the coverslip of a hemocytometer counting chamber.
7. Using an inverted or bright field microscope, with the 10x objective, count the unstained (viable) cells in four of the 1 mm² squares. Calculate the cell number using the formula: cells/mL = cell count/# 1 mm² squares x dilution factor x 10⁴.
   NOTE: For this example, the number of 1 mm² squares counted is four, and the dilution factor is 4.
8. Use the equation C₁V₁ = C₂V₂ to calculate for and prepare a 1 x 10⁵ cell/mL suspension. Prepare 1 mL of cell/mL suspension for every column of the 96-well plate to be filled and 0.75 mL for every chamber of the 4-well chamber slide to be filled according to the plate design.
   NOTE: C₁ = cell count, V₁ = what is being calculated, C₂ = 1 x 10⁵, V₂ = 5.50 mL.
9. Determine V₁, subtract it from the desired 5.50 mL of the total volume to determine the volume of the fresh medium needed to prepare the suspension. Add the volume of cells (V₁) from the original suspension to the volume of fresh medium for a total of 5.50 mL with 1 x 10⁵ cells/mL.
10. Add 100 µL of 1 x 10⁵ cell/mL suspension to each well of the 96-well plate according to the plate design using a multichannel micropipettor and 530 µL to each of the wells of the chamber slide using a micropipette according to the plate design. Incubate overnight.
    NOTE: For this experiment, MSC are plated in columns 1, 2, 3, and 6 of the 96-well plate (Table 1) and wells 1 and 2 of the 4-well chamber slide (Table 2). Therefore, at least 5.50 mL of a 1 x 10⁵ cell/mL suspension is prepared. MΦ at 80% confluence are treated with inflammatory mediators the same day the MSC are seeded in the experimental plates.

3. Activate the MΦ with IFN-γ, Day 1

1. Prepare the activation medium and IFN-γ stock.
   1. For 200 mL of activation medium, add 40 mg of BSA to 200 mL of serum-free high glucose DMEM and sterile filter using a 0.2 µm sterile single-use vacuum filter unit.
   2. To prepare 0.1% BSA/PBS, add 20 mg of BSA to 20 mL of PBS. Vortex to dissolve. Use a 0.2 µm sterile syringe filter to filter into a clean, sterile 50 mL conical tube.
   3. Add 1 mL of the sterile 0.1% BSA/PBS solution to 100 µg of lyophilized IFN-γ to achieve a 100 µg/mL of stock solution. Store in 50 µL aliquots at -20 °C.
2. Activate the MΦ with IFN-γ
   1. Add 2.5 µL of 100 µg/mL of IFN-γ stock for every 1 mL of the medium needed. For every 100 mm dish to be activated, prepare 10 mL of activation medium containing IFN-γ at a concentration of 250 ng/mL.
   2. Aspirate the growth medium from the MΦ cultures and replace it with 5 mL of the activation medium without IFN- γ to rinse.
   3. Aspirate the medium used for rinsing and replace it with 10 mL of IFN- γ supplemented activation medium for the experimental dish and with 10 mL of non-supplemented activation medium for the control. Incubate the cultures for 16-24 h.

4. Isolate the MΦ and prepare the co-cultures, Day 2

1. Remove the activation medium from the MΦ cells and replace it with 5 mL of fresh growth medium. Scrape gently with a cell lifter and collect the cells into a 50 mL conical tube.
2. Count the cells using trypan blue exclusion and hemocytometry as described for MSC in, steps 2.6-2.7.
3. Using the equation in steps 2.8-2.9, prepare two separate suspensions of 2 x 10⁵ cells/mL, one with cells from the control and one with cells from the treated MΦ cultures.
   NOTE: Prepare 1 mL of cell/mL suspension for every column of the 96-well plate to be filled and 0.75 mL for every chamber of the 4-well chamber slide to be filled according to the plate design.
4. Gently aspirate the medium from the experimental wells of the 96-well plate containing MSC. Using a multichannel micropipette, add 100 µL of the treated and the control MΦ cell suspensions in the appropriate experimental wells with and without MSC according to the plate design. Incubate overnight.
5. Gently aspirate the medium from the 4-well chamber slide containing MSC, and, using a micropipette, add 530 µL of MΦ cell suspension to the appropriate wells according to the design. Incubate overnight.

5. Phagocytosis assay, kinetic fluorescent 96-well plate read, Day 3

1. Set the assay parameters
   1. On the day of the assay, turn on the fluorescent plate reader and computer-set the temperature on the system to 37 °C.
   2. Open the system Pro software and check the instrument icon in the upper-left corner to determine whether the instrument is connected to the computer. If the red circle with a line is visible, click on the instrument icon and choose the instrument in the pop-up window to make the connection.
   3. Select New Experiment to access the Plate Set-Up Helper pop-up window. From the Plate Set-Up Helper menu, select Configure Your Acquisition Settings.
   4. Select Monochromator from the settings menu for the optical configuration. Select FL (fluorescence) for the read mode and Kinetic for the read type.
   5. In the same configurations window, under Category, click on Wavelengths, and then set bandwidths to 9 nm for excitation and 15 nm for emission.
   6. Set the Number of Wavelength Pairs to 1. Set the wavelengths LM1 to 510 nm for excitation and to 540 nm for emission.
   7. Continue through the categories and select Plate Type next. Select the option that matches the plate type being used.
      NOTE: It is important that the selection matches the plate type. The read heights are set by the system according to the selection.
   8. Next, select Read Area and highlight the area of the 96-well plate included in the experimental design.
   9. Select PMT and Optics, set PMT Gain to High and Flashes per read to 6. Check the box next to Read from Bottom if using a clear-bottom plate.
   10. Select Timing and set for 10 min intervals over a 70 min period.
   11. Select Shake, check the Before First Read box, and set for 5 s. Check the Between Reads box and set for 3 s. Set shake intensity for both to Low and Linear.
   12. Close the window. When the Plate Set-Up Helper window pops up, select Configure Your Plate Layout. Highlight the BL wells of the plate design and click on Plate Blank.
   13. Highlight each of the experimental rows, click on Add, name the group, and then select a color.
   14. Under Assignment Options below the plate design, select Series, and then define the series in the window and click on OK. Repeat for each group.
2. Prepare the zymosan suspension
   1. While waiting for the system temperature to reach 37 °C, resuspend 1 mg of zymosan particles in 5 mL of the live-cell imaging medium.
      1. Add 1 mL of live-cell imaging medium to the vial containing the particles and collect them into a glass culture tube. Rinse the vial with an additional 1 mL of imaging solution and transfer to the same glass tube to ensure that all the particles are transferred. Add 3 mL of additional imaging solution to achieve a 0.2 mg/mL of particle suspension.
   2. Vortex with quick pulses for 30-60 s. Then, use a probe sonicator to sonicate with 60 quick pulses.
      NOTE: It is very important to create a homogenous suspension of particles and not let the suspension sit too long before adding to the experimental wells. Clumping recurs rapidly. The particle per cell density may need to be optimized depending on the source, treatment, and density of MΦ used. In the studies presented, conjugated zymosan particles were used. However, conjugated E. coli and S. aureus are also available.
3. Add zymosan and read the plate
   1. Aspirate the medium from the experimental wells and rinse 1x with 100 µL of the live-cell imaging solution. Rinse the RBL wells with the live-cell imaging solution as well.
   2. Aspirate the live-cell imaging solution from experimental and RBL wells and replace it with 100 µL of the zymosan particle suspension prepared in section 5.2.
   3. Open the plate tray of the fluorescent reader using the touchpad interface. Set the plate, without the lid, in the tray with the A1 well in the upper-left corner. Close the tray using the touchpad and click on the green Read button in the top menu.
   4. When the read is complete, save the file to the appropriate folder and export the data in a spreadsheet format by clicking on File and selecting Export.
   5. Calculate the mean ± SEM relative fluorescence for each replicate group at each time point (10 min, 20 min, 30 min, etc.) in the spreadsheet (Supplementary File 1) and transfer the data to graphing software.
   6. Use a line graph format to present the data and apply two-way ANOVA (Time x Treatment/MSC as factors) followed by Tukey's multiple comparisons test to determine differences between individual groups.
      ​NOTE: While the 96-well plate read is in progress, prepare the dynamic imaging plate for time-lapse image acquisition. Ensure that the dynamic imaging proceeds with the analysis of one experimental well at a time.

6. Phagocytosis assay, dynamic imaging, Day 3

1. Turn on the imaging system and computer. Double click to open the software. Select the Pro program mode.
2. Check the stage area to ensure no samples are present and nothing is impeding the movement of the stage. Then, click on Calibrate Now.
3. Under the Incubation menu on the sidebar on the right, check the box next to H Unit XL and set it to 37 °C.
   NOTE: Wait until the incubated stage is almost to the temperature before preparing the samples for imaging.
4. Rinse the first experimental well with 750 µL of imaging medium and replace it with 400 µL of the zymosan particle suspension prepared in section 5.2. Leave the remaining wells in the growth medium.
   NOTE: It may be necessary to vortex and sonicate the particle suspension again briefly if it has settled for more than 15 min.
5. Place the slide on the incubated stage of the imaging system. Set a timer for 10 min.
6. Use the imaging software, select Brightfield under the Locate tab, and then click on the eyepiece icon in the system configuration window below. With the 10x objective in place, use the eyepiece and focusing knob to focus on the cells.
7. Select the Acquisition tab, use the Experiment drop-down menu, and then select a set of wavelengths that includes EGFP filter set to accommodate 509 nm excitation 533 nm emission wavelengths of the pH-sensitive dye.
8. When there is ~2 min left on the timer, use the software to change the objective to 20x by clicking on the 20x icon in the objective menu.
   1. Click on the Channels menu, select EGFP filters and use the Exposure menu to set the exposure time to 400 ms to detect the pH-sensitive green fluorophore once it has been enclosed in the phagolysosome.
   2. Click on Live and adjust the focus using the focusing knob.
9. Click on Stop to turn off the light and check the box next to the Time-lapse experimental setting at the top.
10. In the Focusing Strategy menu select Software Autofocus. From the drop-down in the Autofocus menu, select Smart and Coarse settings to reduce the time of exposure to light while the autofocus is running.
11. In the Time-Lapse menu, set the desired number of acquisitions to 30-60 and the interval time to 1 min.
12. After the time-lapse parameters are set and after 10 min of addition of particles to the first well, click on the Start Experiment button to begin acquisition.
13. When the acquisition is complete using the first experimental well, repeat steps 6.4-6.12 for each of the remaining experimental wells.
14. Save all the experiment files with the date and parameters in the title to the appropriate folder.
15. Close the software. Reopen the software in Processing mode and export the files in the MP4 format using the Export menu.