TITLE: Hydrogel Microparticles as Force Sensors: Synthesis and Application in Phagocytosis Studies
AUTHORS: Alvja Mali, Youri Peeters, Mangala Srinivas, Daan Vorselen

[DESCRIPTION]
We provide a protocol for the synthesis of tuneable hydrogel microparticles (HMPs), and their application to measure forces during phagocytosis by macrophages. A specific type of deformabale acrylamide-based HMP, DAAM-particles, are synthesized in batch using a membrane emulsification approach. They then are conjugated with both biologically active molecules and fluorescent labels in a one-pot functionalization procedure. Macrophages are incubated with the functionalized DAAM-particles and imaged by regular confocal microscopy. With a custom image analysis strategy, local DAAM-particle deformations can be quantified with superresolution accuracy (< 50 nm) in 3D throughout phagocytosis. Finally, normal and shear forces can be inferred from the 3D shape of DAAM-particles, revealing the spatial distribution of cellular forces. The entire protocol takes 2-3 days and requires only basic expertise in mammalian cell culture and fluorescence microscopy, and minimal specialized equipment. This protocol can be adapted for studying several other cellular processes, and thereby broadly applied in mechanobiology and immune cell biology.

[STEPS]
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1. Prepare the extrusion equipment


 



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1.2. Flask selection: Check if the flask ensures a proper fit of the SPG module. Allow N2 atmosphere regulation by creating a closed system. Allow addition of the polymerization initiator (AIBN) and pressure release during the process. To simplify workflow, a jacketed flask is recommended but not essential. 

 

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1.3. Set up a temperature control system: Position a water bath next to the set up (without turning it on). 


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3. Preparation of the organic and aqueous phase.
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3.4. In the meantime, mix the components of the aqueous phase. Tested DAAM-particle compositions with specific properties of the resulting particles are given in Table 1.   

 Table 1. Overview of parameters for synthesizing diverse DAAM-particles with different Young's modulus (0.3 – 6.5 kPa) and a (cell-sized) diameter of 9 µm. Typically, a total volume of 12 ml is made. 
 
 
 

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3.5. Place the aqueous phase under a vacuum for 15 min. 


‱ Critical step: Insufficient degassing of the aqueous phase can result in poor particle polymerization.

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6. Measuring DAAM-particles properties.
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6.2. Size
Dilute the DAAM-particles 10-fold in PBS, pH 7.4 and put it on a microscope glass slide with coverslip.
Acquire phase contrast microscopy images of DAAM-particles +(~ 500) with a 40x objective lens.
Analyze the acquired images in IMAGEJ (see note).

 


Calculate the diameter of the microparticles from their respective areas (𝐷 = √(4𝐴/𝜋) where D is the diameter and A the obtained area.
Calculate the coefficient of variation (𝐶𝑉 = 𝑆𝑇𝐷(𝐷)/𝐷)) to evaluate particle batch monodispersity. Where 𝑆𝑇𝐷(𝐷) is the standard deviation and 𝐷 the mean diameter. CV should be ~0.1. 
Calculate the volume/volume (v/v) percentage of the DAAM-particles based on the volume occupied by the spheres (DAAM-particles), multiplied by the concentration of the DAAM-particles. (𝑣/𝑣% = 𝐶 ∗(4/3𝜋∗(𝐷/2)3)/1010 )where C is concentration DAAM-particles/ml, and D the diameter in µm.

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7. Dilute DAAM-particles to a concentration of 5% v/v in PBS (pH 7.4) .
 

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7.1. Take 0.5 mL from the 5% v/v DAAM-particles.

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7.2. Wash the particles twice in 0.5 mL activation buffer by spinning down for 10000 x g, 1 min and thoroughly mixing (by vortexing) in between. 
 

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7.3. Resuspend the washed DAAM-particles in 300 µL activation buffer.

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7.4. Add 5 µLof 10% Tween to the DAAM-particles (0.1 % v/v final concentration)

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7.5. Add 100 µL of 200 mg/mL EDC, freshly dissolved in activation buffer, to the DAAM-particles and thoroughly mix the solution.

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7.6. Add 100 µL of 100 mg/mL NHS, freshly dissolved in activation buffer, to the DAAM-particles and thoroughly mix the solution.

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7.7. Incubate in an end-to-end rotator at Room temperature for 15 min. In the meantime, prepare a new tube with 50 µL of 100 mg/ml BSA solution.


‱ Critical step: During all subsequent resuspension steps (7.8-7.10), vigorously vortex the particles to prevent particle aggregation. When continuing with clustered particles, these will likely be covalently cross-linked and impossible to break up later. Of note, sonication is not recommended as it can break the internal structure of soft DAAM-particles.

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7.8. After incubation, wash the DAAM-particles three times with PBS-T, pH 6.
 

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7.9. Following the final wash, resuspend the DAAM-particles in 225 µL PBS-T, pH 6 and mix the dispersion thoroughly.

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7.10. Add 225 µL of 2x PBS (pH 8.5) to the resuspended DAAM-particles. Mix briefly by pipetting up and down and immediately (one tube at a time) transfer the particles to the BSA solution. 

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7.11. Incubate at Room temperature for 60 min using an end-to-end rotator. In the meantime, prepare a new tube with 5/6-TAMRA cadaverine (0.2 millimolar (mM) final concentration).

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7.12. Add the entire DAAM-particles-BSA solution to the tube containing 5/6-TAMRA cadaverine, mix vigorously, and incubate at Room temperature for 30 min using an end-to-end rotator. 

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7.13. Prepare 250 µL blocking buffer by adding 300 millimolar (mM) ethanolamine to the Tris buffer.

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7.14. Add 250 µL of the blocking buffer to the DAAM-particles solution and incubate at Room temperature for 30 min using an end-to-end rotator.

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7.15. Wash the functionalized DAAM-particles 3 times with PBS-T, pH7.4.

‱ Pause point: After this step, particles can be stored for at least 1 month at 4 °C with 5 millimolar (mM) sodium azide.
 

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8. IgG opsonization

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8.1. Add anti-BSA antibody to DAAM-particles in PBS-T (pH 7.4) to final antibody concentration of 0.1 mg/ml .

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9. Cell seeding

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9.2. Seed 0.75 × 105  J774A.1 cells per well in a 24-well glass-bottom plate and incubate  . 
 

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10. Phagocytosis assay

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10.2. Replace the medium in each well with 200 µL DMEM full containing DAAM-particles (5 × 105 DAAM-particles per well).

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10.3. Centrifuge the plate for 300 x g, 1 min to promote synchronized interactions with cells.

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10.4. Incubate the plate at 37 °C for 5 min-30 min.

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11. Immunostaining 

 

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11.2. Wash wells three times with 200 µL PBS (pH 7.4) to remove fixative solution and unattached DAAM-particles by gently pipetting up and down. 

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11.3. To stain the exposed DAAM-particles surface, replace the medium in the wells with 200 µL of PBS (pH 7.4) containing 4 μg/ml Alexa Fluor-647 donkey anti-rabbit IgG and incubate for 30 min .

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11.4. Wash the wells with 500 µLPBS (pH 7.4) three times.

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11.5. To permeabilize cells, replace the medium in the cells with 200 µL of PBS (pH 7.4) containing 0.2% Triton X-100 solution.

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11.6. Incubate the plate for 10 min.

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11.7. Wash three times with PBS (pH 7.4).

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11.8. To stain cellular actin, add 200 µL of PBS (pH 7.4) containing 0.15 μM Alexa Fluor 488-Phalloidin and incubate for 30 min.

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11.9. Wash three times with 500 µL PBS (pH 7.4).

‱︎︎ ︎ Pause Point: If not imaging immediately, add sodium azide to the wells to a final concentration of 5 mM, wrap the sides of the plate with parafilm, and store at 4 °C. Plates can be stored for a minimum of 1 week.

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12. Imaging

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12.2. Adjust the microscope parameters, including e.g. laser intensity, exposure time, camera EM gain, to obtain properly exposed fluorescence images in four channels: Cellular F-Actin (Phalloidin - Alexa Fluor 488), DAAM-particles (5/6-TAMRA cadaverine), exposed DAAM-particle surface (Secondary antibody - Alexa Fluor 647) using a 60 - 100x, high-NA (~1.4) oil objective. Specifically, ensure appropriate:

Z Range: Capture images with a broad Z range, extending at least 5 µm above and 5 µm below the DAAM-particles.
Step Size: Use a sufficiently fine z-step size (~ 0.15 µm) 
Signal-to-noise ratio (Fig. 4)

 


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12.3. Acquire images of 50 – 100 particles per imaging condition.

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13.1. Open Readme.pdf file and follow the instructions for installing the required Matlab software and packages. 

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13.3. For the next few steps (13.3 - 13.11), run the corresponding code sections by selecting a code section and pressing Ctrl+Enter. A testcase is added to reproduce the output images of the accompanying Plos One manuscript.

Run Read data files: Select your confocal image file in the window that opens. (Testcase: “ProtocolsIO_PlosOne_RAW_IgG_1.3kPa.tif” in the folder “Testdata”)

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11.1. Fix the cells by removing the medium and adding 200 µL of 4% formaldehyde in PBS (pH 7.4) per well.
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12.1. Start the point-scanning or spinning disc microscope according to manufacturer’s procedure.
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1.1. Establish N2 flow from a compressed gas cylinder or a house N2 line. Insert a pressure regulator, shut-off valve and the pressure gauge in the indicated sequence (Fig. 1). Ensure an air-tight fit of the N2 line between the shut-off valve and the top of the SPG module.
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2. SPG Membrane preparation
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2.1.  Choose a SPG membrane with a pore size to synthesize DAAM-particles of your desired diameter (Fig. 2).


 

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2.2. Slowly immerse the membrane in n-dodecane. Continuously allow the membrane to wet itself through capillary forces before submerging it further.
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2.3. Degas the membrane by sonication under vacuum by placing the membrane in a sonication bath while inducing and maintaining a vacuum until no observable bubbles are formed (~5 min). 


‱ Critical step: Inadequate degassing of the membrane can lead to unsuccessful extrusion, as the presence of air within the membrane pores can obstruct the initiation of the extrusion process.

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2.4. Keep the membrane in the n-dodecane until further use.


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3.1. Prepare a 3% v/v solution of Span 80 in hexanes with a total volume of 150 mL. 
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3.2. Stir the organic phase until the Span 80 is completely dissolved.
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3.3. Transfer the organic phase to the (sealable) flask used for polymerization and stir the organic phase 300 rpm under a nitrogen (N2) atmosphere for at least 20 min 

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3.6. Put the aqueous phase under a N2 atmosphere (5 min), ensuring an oxygen-free environment without the need for continuous nitrogen flow.


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4. Setup the SPG module and determine the critical pressure (Pc)
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4.1. Assemble the SPG internal pressure micro kit according to the manufacturer instructions.
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4.2. Take the SPG membrane out of the dodecane and attach it to the internal pressure micro kit extruder, now named SPG module.  
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4.3. Immediately immerse the module slowly into the constantly steering organic phase and ensure a closed system. 
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4.4. Inject 10 mL of the acrylamide mixture (aqueous phase) into the SPG module reservoir under nitrogen pressure using a cannula.  
 

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4.5. Attach the nitrogen line to the SPG module.
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4.6. Determine critical pressure (Pc) following the steps in the note below. If (re)using a membrane with identical pore size to a previously used membrane, these steps can be skipped and the previously determined value can be used instead.

 


‱ Critical step: Identifying the critical pressure and maintaining constant pressure (1.1 – 1.5 times Pc) during extrusion is crucial for establishing monodisperse emulsions. Pressure below Pc preclude flow, and too high pressure (typically 2-3 times Pc) results in highly polydisperse particles. If flow is observed below Pc, there is potentially a leakage between the shaft of the extrusion kit and the membrane, and the aqueous phose is not passing through the membrane, also leading to polydisperse particles.


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5. Emulsification and polymerization
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5.1. After the critical pressure is determined, open the valve and keep the pressure constant during the extrusion of the entire aqueous phase while continuously steering the emulsion (check periodically, about once every hour, to ensure a continuous flow). When extrusion is completed (no gel mixture in SPG module), close the nitrogen flow. Typical time 180 min 
 

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5.2. Carefully remove the SPG module from the emulsion and close the flask containing the emulsion securely.

 

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5.3. Remove the membrane from the module and place it in n-dodecane. Sonicate it for 5 min. Then store the membrane in fresh dodecane.
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5.4. Set the water bath to 62 °Cand start the flow. 
 

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5.5. Weigh out 225 mgof AIBN (1.5 mg/ml final concentration) and add it to the flask. Incubate for 180 min for the polymerization to complete. Periodically release any excess pressure by insertion of a needle. After 3 hours, let the emulsion cool down to room temperature.

‱︎  Pause Point: Polymerized particles are stable. They can be washed and collected the next day.

 

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5.6. Wash the particles three times with an excess of hexanes by spinning down at 300 x g, 5 min.

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5.7. Wash the particle once with an excess of ethanol.

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5.8. Resuspend the particles in 80 mL of PBS and wait approximately 30 min for the particles to rehydrate.
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5.9. Perform two additional washes with PBS by spinning down at 16000 x g, 5 min.

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6.1. Concentration
Dilute the DAAM-particles 100-fold in PBS (pH 7.4) containing 0.1% Tween 20.
Utilize a hematocytometer and a phase contrast microscope to determine particle concentration.
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9.1. Maintain J774A.1 cells in DMEM full (Prepare hiFBS by heating FBS at 56 °C for 30 min).
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10.1. Centrifuge DAAM-particles and resuspend them in DMEM full at 2.5 × 106 DAAM-particles per mL.
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8.2. Incubate at Room temperature for 60 min using an end-to-end rotator.
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5.10. Add sodium azide to a final concentration of 5 millimolar (mM) and store the DAAM-particles at 4 °C.

‱ Pause point: polymerized particles are stable for long term storage (at least 1 year) in PBS 

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8.3.      Wash the functionalized DAAM-particles 3 times with 1X PBS (pH 7.4)
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8.4.      Use directly or store at 4°C in 1x PBS (pH 7.4) with 5mM sodium azide.


‱ Pause Point: after this step, particles can be stored at 4°C for at least 2 weeks with 5 mM sodium azide. 
SECTION: Imaging &amp; data analysis
13. 
 

3D particle reconstruction
Download the MATLAB code for analysing confocal images and deriving particle shape  
14. Force Analysis
Download the Python code for deriving traction forces.
14.2. Open the README.md and follow the suggested installation method to open the script:
Install Anaconda
Open Anaconda Navigator and import ShElastic.yml (located in the ShElastic folder) as new environment
Activate the ShElastic environment
Press home and open jupyter notebook
Forward to the (ShElastic-master -> examples ->) "Case08-Hydrogel_Microparticles_as_Force_Sensors.ipynb"
14.3. Go to Input information and change the following parameters (Testcase: Parameters shown below (Fig. 7))

 



14.4. Go to Kernel (top bar of the screen) and press: "Restart & Run All". This will run the entire code. The output file (output_data.mat) is located in the folder ShElastic (see above) in the subfolder examples.

 

14.5. To visualize and save figures of the analyzed forces, start MATLAB and open the file Usefulforceplots.m (located in DAAMparticle_Shape_Analysis-master folder). Import output_data.mat obtained during the Python analysis.
14.6. Run the code section Plot forces in 2D to visualize the forces on the particle (See accompanying Plos One manuscript, Fig. 2e). Matlab figures can be saved .tiff or .eps files for external viewing.

 

14.1. Create a new folder in ShElastic-master and put the following MATLAB output files in this folder.  (Testcase: ShElastic-master -> testdata):
MyFile_Connectivity (Testcase: Particle01_Connectivity)
MyFile_Coordinates_Cart_smoothed (Testcase: Particle01_Coordinates_Cart_smoothed)
MyFile_Mask_1um_dilated (Testcase: Particle01_Mask_1um_dilated)

MyFile should be replaced with the name corresponding to the output files of MATLAB.
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13.2. Open “ImageAnalysis.m” in MATLAB, and add all the DAAMparticle_Shape_Analysis-master folders and subfolders to the Matlab file path. To do so, first click "Browse for folder" in the upper left corner above the current folder window, then, in the current folder window, rightclick on the DAAMparticles_Shape_Analysis folder (-> Add to Path -> Selected Folder and Subfolders).  


 


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13.7. Run Superparticle edges and triangulate surface. When prompted, use default settings
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13.6. Optional step. Run Deconvolve: Only possible after measuring Point-Spread-Function (PSF) of your microscope. See supplementary Fig. 4 of Vorselen et al., 2020. Usually high-resolution can be achieved without deconvolution, but deconvolution can further improve resolution.
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13.5. Run Threshold the images, watershed and isolate particles. When prompted, use the default settings.

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13.4. Run Get required information from metadata. 
1. Select the DAAM-particle image in the window that opens (see Fig. 6).
2. Enter z-correction factor. Check Vorselen et al., 2020 how to estimate for your own data/microscope. (Testcase: z-correction factor = 1.1)
3. When prompted, enter pixel size values of the images. (Testcase: these values are “PixelSize x and y (µm)” = 0.109 and “Absolute PixelSize Z (µm)” = 0.2) 

 


 

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13.12. To save output, run Save the edge coordinates, triangulation and mask in ImageAnalysis.m to create CSV files with the corresponding data. The IMStats and MPstats that are also saved can be opened in Matlab again to adapt parameters and (re)create figures. 

 


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13.11. To visually inspect the data, open Usefulplots.m and run the following sections:

Plot triangulated surface of particles: This shows the particle 3D shape and a 2D projection of the particle surface (see accompanying Plos One manuscript, Fig. 2d). The particles can be rotated to be inspected from any angle in the matlab figure window.

Plot secondary stain on particle for both IMStain (F-actin) and _ch3 channels to make visualizations of these channels on the particle. The color can be changed at plot options, otherwise use the default options. (Testcase: Grey is actin signal, magenta is secondary antibody signal. See accompanying Plos One manuscript, Fig. 2d). 

Matlab figures can be saved .tiff or .eps files for external viewing.

 

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13.8. Run Determine particle coverage by a secondary signal and select secondary antibody channel (Fig. 6)

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13.10. Run Convert secondary signal to mask, and determine the position of the base of the cup. When prompted, use default settings. This finalizes the analysis.

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13.9. Optional step. Run Determine particle coverage by additional signals. This can help make masks (see 13.10) more accurate in case of uneven antibody staining, and also allows inspecting of F-actin relative to the observed particle deformations. When prompted, select F-actin channel (Fig. 6). (Testcase: select the F-actin channel)