Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

NOTE: All experiments using human biological samples were performed with ethics approval from the Alfred Hospital Human Ethics Committee, Melbourne. All experiments were performed in Class II Biosafety cabinets unless specified. "Prewarmed" refers to reagents warmed to 37 °C in a waterbath.

1. Preparation of Type I Fibrous Collagen Gels: Day 1

1. Prepare polymerized collagen gels by sequentially adding and mixing 35.7 mM NaOH, 0.71 x M199, 4.58 mM acetic acid and 1.71 mg/mL type I fibrous collagen into a 5 mL polystyrene tube as per Table 1.
   NOTE: Ensure that the collagen is well-mixed by gently pipetting up and down 5 times in order to stop the formation of collagen 'pockets' in the gel mixture. Prepare 4-6 gels per test condition for microscopy or 15 gels per test condition for flow cytometry.
2. Once well mixed, aliquot 50 µL of collagen gel mixture into each well of a sterile flat-bottomed 96-well tissue culture plate. Do not use the outside rows and columns, but fill these with 200 µL of 1x Dubecco's phosphate buffered saline (PBS) to protect the gels from desiccation. Incubate plates at 37 °C/5% CO₂ for 2 h to allow the collagen to polymerize.
   Note: Place the plates directly on clean metal racks in the incubator to ensure even heat distribution.
3. Following incubation, overlay the gels with 150 µL of 1x supplemented M199 (see Table of Materials) and incubate for 5 days until use.

2. Expansion of Stored HUVEC: Day 1

1. Label and coat a 10 cm diameter Petri dish with 1 mL of 50 µg/mL fibronectin diluted in 1x PBS and incubate at room temperature for 10 min.
2. Thaw aliquots of cryopreserved primary human umbilical vein endothelial cells (HUVEC, 1.0 x 10⁶ cells) in 10 mL M20.
   NOTE: HUVEC should be prepared as previously described²¹ and used at a low passage number (< passage 4). HUVEC may be replaced with human coronary artery endothelial cells here.
3. Resuspend HUVEC in 10 mL M20 and add to fibronectin coated dish, aspirating excess fibronectin prior to addition of cells, and culture to confluence (approximately 5 days), replacing the media on Day 3.

3. Culturing HUVEC Monolayer on Collagen Gels: Day 5

1. On Day 5, detach HUVEC by aspirating culture supernatant from the Petri dish and washing away serum-containing media with 10 mL of serum-free M199.
2. Add 5 mL 0.05% trypsin/0.53 EDTA in M199 to HUVEC and incubate for 1-2 min at room temperature, shaking gently until the cells detach.
3. Once detached, quickly rinse Petri dish with 5 mL M20 and transfer the media containing cells to a 10 mL tube.
4. Centrifuge samples at 300 x g for 5 min at room temperature, aspirate supernatant, resuspend cells in 200 µL of M20, and count cells using a hemocytometer.
5. Resuspend the cells to 2.0 x 10⁵ cells/mL in M20, aspirate the M199 on the gels from the 96-well plate (step 1.3) and add 100 µL of resuspended HUVEC (2.0 x 10⁴ cells) to each collagen gel prepared above (step 1.2). Culture plates for a further 3 days at 37 °C/5% CO₂.
   NOTE: The integrity of the HUVEC monolayer may be confirmed after 3 days by silver nitrate staining²² or immunohistochemistry for tight junction proteins²³ at this stage. Additional gels must be prepared for this purpose.

4. Activation of HUVEC Monolayer and Isolation/Activation of Monocytes for Transmigration: Day 8

1. On Day 8, activate each HUVEC monolayer prior to the addition of monocytes by aspirating the M20 media overlaying the gels and adding 100 µL of 10 ng/mL human TNFα in M20 per gel. Incubate at 37 °C/5% CO₂ for 4 h.
   NOTE: Non-activated HUVEC conditions may also be included if required as controls.
2. During the 4 h HUVEC activation step, isolate monocytes from PBMCs using magnetic bead techniques to negatively select for cells as per the manufacturer's instructions. A minimum of 6.5 x 10⁶ PBMCs should be used at this step in order to reliably recover 3.0 x 10⁵ monocytes required for one condition, as monocytes typically account for approximately 10-15% of PBMCs.
   NOTE: To evaluate the effect of monocyte activation prior to monocyte transmigration and foam cell formation, monocytes can be activated at this stage. Monocytes can be isolated from thawed PBMCs if required (i.e., stored patient samples). Monocyte subsets isolated by FACS sorting can be prepared for addition to gels (Figure 1).

5. Transmigration of Primary Human Monocytes: Day 8

1. To measure monocyte transmigration, remove TNFα-containing media and wash gels twice with 100 µL M199 by adding and removing media. Following washing, add 2.0 x 10⁵ freshly isolated or thawed and washed cryopreserved PBMCs or 5.0 x 10⁴ freshly isolated monocytes or purified monocyte subsets to HUVEC monolayers in 100 µL M20. Incubate for 1 h at 37 °C and 5% CO₂ to allow the forward transmigration of monocytes into the gel. It is best to allow 6 wells for every experimental condition examined.
   NOTE: To evaluate the effect of autologous serum on transmigration and foam cell formation, incubate cells with M20 containing the desired concentration of heat-inactivated donor serum and compare to conditions with a pooled human serum control. Leukocytes other than monocytes may be added at this stage. If investigating the impact of specific lipids/lipid species (e.g., HDL, oxHDL), perform all following steps with serum-free media containing 20-50 µg/mL lipids.
2. After 1 h of forward transmigration, collect the non-transmigrated cells by washing gels twice with 100 µL of prewarmed 1 mM EGTA in 1x PBS, and once with 100 µL M199 (same centrifuge conditions), pooling the supernatants from each well of the same experimental condition (usually 6 wells) into a 1.5 mL microcentrifuge tube on ice. Centrifuge the non-transmigrated cells at 4 °C, 300 x g for 5 min and resuspend in 30 µL 1x PBS.
3. Count the cells collected in the supernatant to determine the number of forward transmigrated cells.
4. The percentage of forward transmigrated cells is determined as follows:
   
5. Cover the washed gels containing transmigrated cells with 100 µL M20 and incubate for a further 48 h.
6. After 48 h, collect the supernatant and wash non-transmigrated cells twice with 100 µL prewarmed 1 mM EGTA/PBS, collecting and pooling the supernatant from each condition as in step 5.2.
   NOTE: The phenotype of reverse transmigrated cells may be tested on these cells following standard flow cytometry staining protocols.
7. Centrifuge cells at 4 °C, 300 x g for 5 min and resuspend cells in 30 µL 1x PBS. Count cells and determine the viability via trypan blue staining.
8. Determine the percentage of reverse transmigrated cells by using the following equation:
   
   NOTE: Accounting for the total number of forward transmigrated cells gives a more accurate indication of reverse transmigration as it is unlikely forward transmigration will be 100%.
9. For analysis by microscopy, fix the gels by adding 100 µL 2% formaldehyde (final concentration) to each well, cover the plates in aluminum foil and store at 4 °C until analysis. For flow cytometry, do not fix the cells at this stage. See the protocols below for microscopy (section 6) or flow cytometry (section 7) analysis.
   CAUTION: Formaldehyde is toxic and corrosive; use personal protective equipment (PPE). Care should be taken when adding formaldehyde, however, this step does not need to be performed in a fume hood due to the low concentration used.

6. Quantitation of Foam Cells and Macrophages by Microscopy (Oil-Red O Stain)

1. Remove the formaldehyde and wash the gels with 100 µL of 50% (v/v) methanol for 5 min and then 100 µL 78% (v/v) methanol for 15 min.
   NOTE: The staining steps may be performed on the laboratory bench and not in a Class II Biohazard cabinet.
2. Stain for lipid droplets by adding 100 µL of freshly prepared 0.2% (w/v) Oil-Red O (see Table of Materials for details) for 1 h at room temperature.
3. Remove excess stain by washing the gels 4 times with 100 µL of 78% (v/v) methanol, aspirating and discarding the supernatant after each wash.
4. Counterstain for 15 min at room temperature with 100 µL of Giemsa, diluted 1:10 in distilled water.
5. Aspirate the Giemsa stain and wash gels once with water.
6. To detach the gels from the plate, rim the wells using a 21 G needle, with the bevel facing outwards, around the edge of the gel.
7. To mount the gels on a microscope slide, punch two holes (6.35 mm diameter) in a 2.54 cm x 1.5 cm strip of double-sided tape. Fix the tape to a standard microscope side and remove the protective coating from the top.
   1. Add a drop of water to the holes in the tape using a transfer pipette. Using tweezers, gently transfer gels to the holes in the tape and cover with a size 1.5 glass coverslip. Press gently on the coverslip to adhere it to the tape.
8. Examine with differential interference contrast bright-field microscopy using 40X objective on an inverted microscope.
   1. Bring the HUVEC monolayer into focus at 40X magnification and scroll 'into' the gel, counting macrophages/foam cells throughout the entire depth of the gel. Repeat for three distinct fields of view located at similar distances from the edge of the gel in order to minimize possible edge effects. This will ensure gel depth is consistent between counting regions.
      NOTE: Score cells in the gel as either foam cells (defined as cells containing >1/3 of their cytoplasm as Oil-Red O stained lipid droplets) or macrophages within 2 h of mounting on slides (Figure 2). Foam cell formation is expressed as the percentage of foam cells relative to the total number of migrated cells and is expressed as the median counts in 3 fields of view examined for 6 gels per condition, therefore a median of 18 measurements per condition. An example of raw cell counts from a typical experiment is shown in Table 2.
      NOTE: Classifying a cell as a foam cell vs macrophage by microscopy is subjective and consequently can be investigator-dependent. In clinical studies, where experiments are performed over an extended period of time, it is important for all counting to be performed blinded by a single investigator. Examples of foam cells and macrophages in gels are provided in Figure 2.

7. Analysis of Transmigrated Cells by Flow Cytometry

1. To phenotypically characterize foam cells and macrophages following transendothelial migration, digest the gels by adding 100 µL of 37 °C prewarmed 1 mg/mL collagenase D diluted in M199 to each well and incubate for 20 min at 37 °C/5% CO₂.
   NOTE: Place the 96-well plates directly on clean metal racks in the incubator to ensure even heat distribution.
2. Following incubation, macerate the gels using a 200 µL pipette tip and incubate for a further 20 min at 37 °C/5% CO₂.
3. Once fully digested, filter and pool the digested replicate gels through 35 µm nylon mesh capped FACS tubes placed on ice, and wash once with FACS wash (see Table of Materials) at 4 °C, 300 x g. Resuspend the cells in 100 µL of FACS wash.
4. Incubate the resulting cells with fluorophore-conjugated antibodies specific for CD45, a live/dead cell marker and surface/intracellular phenotypic or functional markers of interest using standard flow cytometry protocols.
   NOTE: Prepare control tubes for compensation using the appropriate fluorophores and Ig compensation beads. Extracted cells may also be collected onto glass slides for immunofluorescence microscopy by cytospinning. Alternatively, we have successfully collected cells using this protocol for assessment via imaging flow cytometry.
5. Acquire cells using a flow cytometer, and perform compensation as required.
   NOTE: As foam cells/macrophages may not form individual populations using light scatter properties and may differ considerably in size and shape, set liberal forward scatter (FSC) and side scatter (SSC) gates in order to capture all migrated cells as shown in Figure 3A.
6. Following acquisition, perform the data analysis using flow cytometry software. To identify migrated cells, first select cells as negative for the live/dead marker as shown in Figure 3B. Next, perform a single-cell discrimination by creating a tight gate around the cells shown on a SSC-area vs SSC-height plot.
7. Select CD45⁺ cells and gate the major population of migrated cells present in a FSC/SSC plot as these are the macrophages/foam cells.