A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

All methods described here have been approved by The Korea Advanced Institute of Science and Technology Institutional Animal Care and Use Committee (IACUC), KA2016-08.

1. Synaptosome Purification

NOTE: These procedures are adapted from a previously published paper¹⁹ with several modifications to improve the yield of purified synaptosomes (Figure 1).

1. Prepare discontinuous density gradient media.
   1. Place the following on ice: 3%, 10%, and 23% density gradient solutions in gradient buffer (bring to 250 mL of water with 109.54 g of sucrose, 606 mg of Tris, and 5 mL of 0.2 M EDTA, pH 7.4); homogenizing buffer (add 25 mL of 4x gradient buffer and 500 µL of 50 mM DTT into 74.5 mL of water); homogenizer mortar and pestle.
      NOTE: Density gradient solution and preparation are presented in the Table 1. To prepare 3%, 10%, and 23% density gradient solutions, use raw density gradient solution.
   2. Prepare six ultra-clear centrifuge tubes per three adult mice.
   3. Add 3 mL of 23% density gradient solution on the bottom of each centrifuge tube with a 1-mL pipette. Then, slowly make a layer with 3 mL of 10% density gradient solution on top of the 23% density gradient solution with a pasture pipette and 1-mL pipette. Finally, add 3 mL of 3% density gradient solution on top. In this step, use caution to avoid introducing bubbles.
   4. Cover the centrifuge tubes with plastic wrap and keep the tubes on ice.
2. Precool a high-speed centrifuge and ultracentrifuge to 4 °C.
3. Sterilize surgical equipment (operating scissors, Castroviejo spring scissors, and curved forceps) with 70% ethanol. Prepare a small glass beaker with 25 mL of homogenizing buffer on ice and weigh the beaker.
4. Extract the brains from three adult (approximately 12 weeks old) male mice.
   1. Dislocate the cervical vertebra and cut the neck with operating scissors. Peel off the skin of the head and skull along the midline using operating scissors and curved forceps. Excise the olfactory bulbs and cerebellum with Castroviejo spring scissors.
5. Put the remaining brains into the beaker with curved forceps and weigh the brains. Rinse the brains several times with ice-cold homogenizing buffer to remove blood on the surface of brains.
   NOTE: Previously, 9 mL of homogenizing buffer to 1 g of brain tissues was recommended.
6. Add 4 mL of homogenizing buffer and 1 g of brain tissues to the homogenizer mortar. While homogenizing the brains, add homogenizing buffer up to 8 mL.
7. Divide the homogenate into two 50-mL high-speed centrifuge tubes. Wash the mortar with 1 mL of fresh homogenizing buffer and add it to the tubes.
   Caution: Proceed through steps 1.3-1.7 as quickly as possible. Less than 20 min is recommended.
8. Centrifuge the homogenates in high-speed centrifuge tubes at 1,000 x g for 10 min at 4 °C with slower brakes.
   NOTE: Set the deceleration rate (brake) at two or three when the maximum rate of deceleration is nine.
9. Carefully add the supernatant to a new 50-mL tube and add up to 12 mL of homogenizing buffer.
10. Carefully and slowly pipette 2 mL of diluted supernatant over the 3% density gradient solution with a 1-mL pipette and place the tubes on ice.
11. Centrifuge at 31,000 x g for 5 min at 4 °C with no brakes.
    NOTE: Set the deceleration rate at one (zero) or coast.
12. Place the tubes on ice and collect the synaptosome fraction between the 23% density gradient solution and 10% density gradient solution with a 2-mL pipette into a 50-mL tube. Add ice-cold sucrose/EDTA buffer up to 80 mL and divide it into four 50-mL high-speed centrifuge tubes.
    NOTE: See Figure 3 for fractionated synaptosomes with labeled gradients.
13. Centrifuge at 20,000 x g for 30 min at 4 ˚C with fewer breaks.
    NOTE: Set the deceleration rate at two or three when the maximum rate of deceleration is nine.
14. Carefully remove the supernatant with a 1-mL pipette, resuspend the synaptosome pellet with 1 mL of isotonic physiological buffer (140 mM NaCl, 3 mM KCl, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 10 mM HEPES, and 10 mM glucose, pH 7.4)²⁰. Add 1 mL of 10% DMSO in isotonic physiological buffer (final concentration of DMSO: 5%) and collect the synaptosomes into one 50-mL high-speed centrifuge tube.
15. Aliquot 1 mL into 1.5-mL tubes. Measure the protein concentration of the synaptosomes using the Bradford assay.
16. Quickly freeze the tubes and store the tubes in a -80 °C deep freezer until pH indicator conjugation.

2. pH Indicator Conjugation

1. Centrifuge 1.5-mL tubes with synaptosomes at 21,092 x g for 3-4 min at 4 °C.
2. Remove the supernatant, add 200 µL of 0.1 M Na₂CO₃ and mix well by pipetting.
   NOTE: Na₂CO₃ was added to raise the pH as succinimidyl ester most efficiently reacts with primary amines at a slightly alkaline pH.
3. Add 2 µL of pH indicator to the tube and gently vortex.
   Caution: Use 1 µL of pH indicator per 0.3 mg of synaptosome solution.
4. Minimize light exposure by covering the tubes with aluminum foil and incubate them for 1-2 h at room temperature (RT) in a twist shaker with gentle agitation at 30-40 rpm.
5. Stop agitation and add 1 mL of DPBS.
6. Centrifuge the tubes at 21,092 x g for 1-2 min.
7. Remove the supernatant and add 1 mL of DPBS to resuspend the pellet by gentle pipetting.
8. Repeat steps 2.6-2.7 at least seven times to remove unbound pH indicator.
9. Remove the supernatant and resuspend the pellet with 200 µL of DPBS with 5% DMSO.
   NOTE: At this point, all synaptosomes are non-functional. We have confirmed that phosphatidylserine (PS) has been exposed to the outer membrane of synaptosomes, which functions as an "eat-me" signal (Figure 4).

3. Astrocytes and Microglia Purification

NOTE: This protocol for astrocyte purification is adapted from a previously published paper²¹. In this protocol, the purification of rat astrocytes and microglia is described. Specific procedures for purifying mouse astrocytes and microglia are also described in the notes.

1. Day before purification
   1. Prepare pre-coated cell culture dishes and solutions.
   2. Prepare five 145 mm x 20 mm Petri dishes with 25 mL of 50 mM Tris-HCl (pH 9.5). Place primary or secondary antibody treatment in the Petri dishes separately as described below. Incubate the dishes in the freezer overnight.
      BSL-1 dish: 20 µL of 5 mg/mL BSL-1 (Griffonia simplicifolia lectin)
      Secondary-only dish: 60 µL of 2.4 mg/mL goat anti-mouse IgG+IgM (H+L)
      CD45 dish: 60 µL of 2.4 mg/mL goat anti-mouse IgG+IgM (H+L)
      O4 dish: 60 µL of 2.4 mg/mL goat anti-mouse IgM (µ-chain specific)
      Itgb5 (Integrin β5) dish for rat astrocytes: 60 µL of 2.4 mg/mL goat anti-mouse IgG+IgM (H+L)
      NOTE: To attach secondary antibody to the hydrophobic surface of the Petri dish, slow attachment at low temperatures is recommended. However, it is possible to coat the Petri dish with secondary antibody for 2 h at 37 °C. Antibodies for astrocyte panning are used differently depending on the animal species; for example, HepaCAM dish for mouse astrocytes: 60 µL of 2.4 mg/mL goat anti-mouse IgG+IgM (H+L)
2. Day of Purification
   1. Prepare for immunopanning.
   2. Prepare 50-mL tubes and add stocks to the tubes. The detailed stock solution preparation is presented in the Table 1.
      1. Prepare Tube 1 (Enzyme): 22 mL of enzyme stock (Enzyme stock: 1x Earle's Balanced Salt Solution (EBSS)+0.46% D(+)-glucose+26 mM NaHCO₃ and_{0.5 mM EDTA).}
      2. Prepare Tube 2 (Low Ovo): 42 mL of inhibitor stock (Inhibitor stock: 1x EBSS+0.46% D(+)-glucose+26 Mm NaHCO₃).
      3. Prepare Tube 3 (High Ovo): 10 mL of inhibitor stock.
   3. Attach 0.22-µm syringe filters to the tips of 2-mL pipettes connected to a CO₂ (95% O₂ and 5% CO₂) tank. Bubble CO₂ into Tubes 1-3. Stop bubbling when the solutions turn orange.
   4. Complete the solutions.
      Caution: Complete and incubate the solution in Tube 1 at 34 °C for 15 min before using for enzyme activation.
      1. Prepare Tube 1 (Enzyme): 22 mL of enzyme stock + 180 U of papain + 0.004 g L-cysteine.
      2. Prepare Tube 2 (Low Ovo): 42 mL of inhibitor stock + 3 mL of Low Ovo + 200 µL of DNase.
      3. Prepare Tube 3 (High Ovo): 10 mL of inhibitor stock + 2 mL of High Ovo + 20 µL of DNase.
      4. Prepare Tube 4 (0.2% BSA): 19 mL of 1X DPBS + 1 mL of 4% BSA + 8 µL of DNase.
      5. Prepare Tube 5 (0.02% BSA): 45 mL of 1X DPBS + 5 mL of Tube 4 + 50 µL of DNase.
   5. Preheat a water bath and heat block at 34 °C.
3. Extract the rat cortex.
   1. Sterilize surgical equipment with 70% ethanol and prepare 100-mm Petri dishes with DPBS.
   2. Prepare an acrylic box. Put two to three layers of tissues in the bottom of the box and add 1 mL of isoflurane into the box.
   3. Anesthetize pups for 20-40 s in the box and confirm anesthetization by foot pinch with forceps. Expose the heart²² and perfuse pups using a 10-mL syringe with DPBS.
      NOTE: Perfusion is used to avoid blood cell contamination during the microglia panning step.
   4. Cut the pup's upper cervical line of the neck with operating scissors and incise the head skin along the midline. Make an incision on the skull along the midline and open the skull with curved forceps.
   5. Excise the cerebellum with Castroviejo spring scissors. Take out the remaining brain and place it in a 100-mm Petri dish filled with DPBS.
   6. Remove other areas such as the midbrain and hippocampus from the cortex with Castroviejo spring scissors under the microscope. Remove the meninges with fine forceps.
   7. Transfer the cortex into a new 60-mm dish and remove the remaining DPBS in the dish. Chop the tissue with a No. 10 blade.
4. Dissociate the cortex into a single cell suspension
   1. Pour the filtered Tube 1 solution into a 60-mm dish and add 50 µL of DNase1. Swirl the solution in the dish.
      NOTE: DNase is used to inhibit aggregation of DNA from ruptured cells.
   2. Place the dish into a 34 °C heat block and cover it with a lid that has a hole in the middle. Use a soldering iron to make the hole in the lid of the 60-mm Petri dish.
   3. Connect 22-µm syringe filters to a CO₂ tank tube and place the filter tip into the hole.
   4. Incubate the dish at 34 °C for 45 min. Swirl the solution in the dish every 10 to 15 min.
5. Finish preparation of the panning dishes.
   1. Remove the panning dishes that have been coated with secondary antibodies from the freezer a day ahead of time. Leave the BSL-1 dish and secondary antibody-only dish at RT.
   2. Wash the other dishes three times with 20 mL of DPBS.
   3. Pour an adequate amount of 0.02% BSA into the dishes. Place specific primary antibodies into the corresponding dishes:
      CD45 dish: 12 mL of 0.02% BSA + 20 µL of 0.5 mg/mL Rat anti-mouse CD45
      Itgb5 dish: 12 mL of 0.02% BSA + 20 µL of 0.5 mg/mL Itgb5
      O4 dish: 8 mL of 0.02% BSA and 4 mL of O4 antibody
      NOTE: For mouse immunopanning, use mouse anti-rat CD45 (0.5 mg/mL) for microglia and HepaCAM (0.5 mg/mL) for astrocytes.
   4. Prepare 30 mL of 30% FBS with neurobasal media and 10 mL of 1X EBSS. Warm up both solutions in a 34 °C water bath.
6. Inhibit the papain reaction.
   1. Transfer the papain-treated tissues to a new 50-mL tube. Wait for the tissues to settle. Aspirate liquid by suction.
   2. Add 4 mL of Low Ovo solution to the tube and swirl the solution to wash the cells.
   3. Repeat steps 3.6.1-3.6.2 three times to stop the enzyme reaction.
7. Triturate the cortex tissues.
   1. Add 6 mL of Low Ovo to the tub. Aspirate and release the tissues quickly with a 5-mL pipette.
      Caution: Do not introduce bubbles.
   2. Prepare a new 50-mL tube and add 5 mL of Low Ovo. Collect single cells in a new tube.
   3. Repeat steps 3.6.1-3.6.2 two times and change the 5-mL pipette to a 1-mL pipette.
   4. Repeat trituration until more than 95% of the tissues are not visible.
   5. Make a layer of High Ovo solution from tube 3 with a 10-mL pipette below low Ovo-containing dissociated cells.
   6. Centrifuge at 190 x g for 6 min with few brakes.
      NOTE: Set the deceleration rate at one or two when the maximum rate of deceleration is nine.
8. Filtrate single cells.
   1. Carefully aspirate the supernatant by suction, then resuspend the pellet with 3 mL of 0.02% BSA solution with a 1-mL pipette.
   2. Add 0.02% BSA solution up to 9 mL.
   3. Prepare a new 50-mL tube and 20-µm cell strainer on top of the tube. Wet the cell strainer with 1 mL of 0.02% BSA solution.
   4. Transfer the single cell suspension into the cell strainer. Filter 1 mL at a time.
   5. Wash the cell strainer with 3 mL of 0.02% BSA solution. Do not make more than 15 mL of the filtered single cell suspension.
   6. Incubate the tube in a 34 °C water bath for 45-60 min for cell recovery. The cell recovery step allows for relocalization of cell surface proteins to the membrane.
9. Perform panning of the cells.
   1. Wash the CD45 panning dish three times with 20 mL of DPBS. Wash every dish three times with 20 mL of DPBS immediately before use.
   2. Pour the single cell suspension into the CD45 panning dish. Leave the dish for 28 min and shake the dish for 14 min. To remove unbound cells from the bottom, carefully shake the dish.
   3. Transfer the cell suspension to the secondary-only dish. Wait for 20 min and carefully shake the dish for 10 min. To obtain microglia from the CD45 dish, go to step 3.9.2.
   4. Transfer the cell suspension into the BSL-1 dish. Leave the dish for 10 to 12 min.
   5. Transfer into the O4 dish. Wait for 20 min and shake for 10 min.
   6. Transfer into the Itgb5 dish. Leave the dish for 40 min and gently shake every 10 min.
      NOTE: For mouse panning, transfer the cell suspension into the HepaCAM dish.
10. Detach cells from the dish.
    1. Mix 400 µL of trypsin (30,000 U/mL in EBSS) with preincubated 8 mL of 1X EBSS.
    2. Pour the detached cell suspension in the dish into a waste bucket.
    3. Wash the dish eight times with DPBS.
    4. Pour 8 mL of EBSS with trypsin into the dish.
    5. Incubate the dish for 5 to 10 min in the 37 °C incubator. Incubate for 10 min for the CD45 dish and 5 min for the Itgb5 and HepaCAM dishes.
    6. Tap the dish and observe the dish under a microscope.
       NOTE: If most of the astrocytes have not detached, put the dish back into the incubator for another 5 min.
    7. Pour 10 mL of 30% FBS into the dish to neutralize trypsin.
    8. Pipette up and down in the entire dish to detach astrocytes or microglia.
    9. Transfer the solution into a 50-mL tube.
    10. Add 10 mL of 30% FBS and 200 µL of DNase into the dish. Detach the cells again. DNase is used to inhibit aggregation of DNA from ruptured cells.
    11. Transfer the solution into the tube. If cells remain in the dish, add another 10 mL of 30% FBS and detach the cells again.
11. Plate the cells.
    1. Centrifuge the tube at 213 x g for 10 min.
    2. Aspirate the supernatant and resuspend the cell pellet with media.
       NOTE: Use immunopanned astrocyte basic media (IP-ABM) for astrocytes and retinal microglia basic media (MBM) with immunopanned astrocyte-conditioned media (IP-ACM) for microglia. The IP-ABM and MBM compositions are presented in the Table of Materials.
    3. Count the cells with a hemocytometer.
    4. Plate the astrocytes and microglia separately in PDL-coated plates.
       NOTE: Prepare the PDL-coated plate before use. To prepare PDL-coated plates, add 50 µL of 1 mg/mL poly-D-lysine into 5 mL of distilled water and pour an adequate amount of solution into the well plate/dish, and wait for 30 min. Wash the plate/dish three times with distilled water.
    5. Incubate the plate. Change half of the media every 7 days for astrocytes and 3 days for microglia.

4. Collect IP-ACM

1. Prepare a 100-mm dish when astrocytes are overly confluent.
2. Discard the media and wash the dish three times with 10 mL of DPBS.
3. Pour 15 mL of low protein media into the dish.
   NOTE: The low protein media composition is presented in the Table of Materials.
4. Incubate the dish for 7 to 10 days in the 37 °C incubator.
5. Collect and concentrate the media with 10k (or 30k) centrifugal filter tubes.
6. Centrifuge the tubes at 851 x g at 4 °C.
   NOTE: Centrifuge the tubes until the remaining media is less than 1 mL.
7. Collect concentrated media (IP-ACM) into a new 1.5-mL tube.
8. Measure the concentration of IP-ACM using quantitative analysis

5. Phagocytosis Live-imaging Assay (Figure 2)

1. Prepare a 24-well plate (or any plate/dish that is prepared for live-imaging) in which astrocytes are confluent (generally, 7 to 10 days of incubation after the purification is ideal for stabilizing the cells).
2. Remove the media in each well and wash the wells with 1 mL of DPBS, three times. To minimize air exposure, wash the wells quickly.
3. Add 300 µL of IP-ABM with 5 µL of pH indicator-conjugated synaptosomes and additional factors such as IP-ACM that can modulate glial cell phagocytosis.
4. Incubate the plate in a CO₂ incubator for 40 min. This step allows the pH indicator-conjugated synaptosomes to settle to the bottom of the plates.
5. Remove the media with unbound pH indicator-conjugated synaptosomes and wash each well with 1 mL of DPBS, three times.
   Caution: Do not wash harshly. To minimize air exposure, wash the wells quickly.
6. Add 500 µL of IP-ABM with additional factors to test into the wells.
7. Take the plate to a live-imaging instrument and select positions. Adjust focus, exposure time, brightness, and LED power.
   NOTE: The settings for exposure time, brightness, and LED power are variable depending on fluorescence intensity and the purpose of the experiment. In the live-imaging assay with pH indicator-conjugated synaptosomes, we usually set those values as follows: exposure: ~ 150-200 ms, brightness: 15, and LED power: 4-6.
8. Set the image format, time interval, and the total number of cycles for live-imaging. Set the time interval to 1 or 2 h depending on how many positions are selected. 2 hour intervals are recommended when more than 150 positions are selected for live-imaging.
9. Start the live-imaging experiments.
10. Analyze the data.
    1. Open the Fiji program.
    2. Import an image sequence. Convert images to 8-bit grayscale.
    3. Perform background subtraction with a rolling bar radius of 50 pixels.
    4. Start time series analyzer V3 plugins.
       NOTE: The address for time series analyzer V3 plugins is presented in the Table of Materials.
    5. Drag the region of interest (ROI) in the image and click add in the ROI manager.
    6. Click "Get total intensity" in the Time series V3_0.
    7. Save the results and integrate the data.