Protocol for assessing phagocytosis activity in cultured primary murine microglia

Summary In this protocol, we describe steps to assess inflammation-induced cell response in cultured primary murine microglia through the analysis of fluorescent bead phagocytosis. We detail primary murine mixed glial cell culture preparation followed by microglia-specific isolation. Further, we describe treatment with lipopolysaccharide (LPS) to induce phagocytosis of fluorescent beads, followed by quantitative analysis using fluorescent imaging and Fiji – ImageJ software. For complete details on the use and execution of this protocol, please refer to Parrott et al.1


Total 10 mL
Filter using a 0.2 mm filter. Prepare 1 mL aliquots. Store at À20 C, stable for 6 months.

Total 50 mL
Store at À20 C, stable until expiration date shown on label, thaw at 4 C before plating. Note: Warm 650 mL 13 PBS to 55 C and add 40 g PFA. Cover with aluminum foil to prevent PBS from evaporating and stir until solution becomes clear while maintaining the temperature at 55 C. Allow PFA solution to cool to 18 C-26 C, then adjust volume to 1 L with 13 PBS. Filter using a 0.2 mm filter and aliquot PFA into 50 mL conical tubes. When protected against light, filtered 4% PFA aliquots can be stored at À20 C for several years, 4 C for several weeks, or 18 C-26 C for 1-2 weeks.
CRITICAL: When dissolving the PFA into 13 PBS, make sure the temperature remains constant at 55 C. Deviations can prevent complete dissolution of PFA or can interfere with the chemical structure. Prepare PFA under a fume hood.
Note: If the PFA fails to dissolve when warmed, add 1 N NaOH dropwise to the solution until the solution is clear. After filtering the solution, use a pH probe to adjust solution pH to 6.9 with filtered 1 N NaOH or filtered 1 N HCl. Note: This set-up is intended for the dissection of 2-4 pups for one culture. If preparing multiple genotypes at once, repeat this set-up for each genotype and label as necessary.

Dissection and tissue collection
Timing: 2 h Note: Prior to beginning dissection, 10% FBS/DMEM/PS media should be prepared (see materials and equipment) and an aliquot of about 20 mL should be incubated at 37 C.
Note: A video demonstrating cortical dissection similar to the method described below has been previously published. 4 Please note this video example uses embryonic pups as opposed to PND 2 pups. 4. Decapitate the pup to separate the head from the body.
a. Spray the head of the pup with ethanol and grip back (dorsal side) of the body tightly to pull the arms outward. b. Flip the pup so that its stomach (ventral side) is facing up. c. Quickly decapitate the pup with sharp scissors. 5. Separate the brain from the skull to prepare for brain dissection.
a. Gently remove the skin from the top of the skull (dorsal side) by pulling skin away from the eye sockets with forceps. b. Cut open the skull along the midline. Begin at the brainstem opening and continue to the anterior portion of the skull (near eye sockets). c. Peel the two sides of the skull back from the midline cut until the skull has been removed from the top of the brain tissue. d. Use a small scoop tool to remove the brain from the base (inferior portion) of the skull. e. Place the brain in the petri dish containing HBSS-H and keep on ice. 6. Remove the meninges, cerebellum/brainstem, olfactory bulbs, and hypothalamus with forceps ( Figure 1A). 7. Using a dissecting scope, dissect out the hippocampus and striatum to isolate the cortex ( Figures 1B and 1C). Make sure to keep the tissue/HBSS-H cold. a. With the ventral side of the brain exposed and the anterior of the brain to the top, use forceps gently roll the cortical layer up to expose the hippocampus. b. From the corners created where the two cuts meet at midline, roll back the ventral part of the cortex away from midline to expose the hippocampus. c. Invert the brain (posterior of the brain to the top) and slide curved forceps under the hippocampus to find the lateral edge. d. Disconnect the hippocampus on both lateral sides from the cortex underneath. Also, separate the hippocampus from the tissue that is on the inferior edge.

OPEN ACCESS
e. Identify the striatal tissue (inferior to the hippocampus) and use the curved forceps to remove the striatum as well. Be careful to only remove the superficial portion of the tissue as there is more cortex underneath the striatum. 8. Collect the cortical tissue pieces from the petri dish and place into the designated 15 mL conical tube containing 10 mL HBSS-H ( Figure 1D).
Note: (Optional) The collected tissues can be cut into small pieces with forceps or razor blades before placing into the conical to aid the trypsinization process.  Note: During dissection, tail tissue can be collected from pups and can be used for genotyping to confirm assumed genotype.
Note: If planning to dissect many pups (greater than 6), consider performing multiple dissections so that the tissue remains on ice for no longer than 60 min.

Mixed glial cell culturing
Timing: 1 h CRITICAL: Glial culturing should be conducted in a sterile environment, ideally a cell culture hood.
Note: DNase should be prepared before beginning culture (see materials and equipment). 10. Aspirate (using a vacuum suction system) HBSS-H from the conical tube containing the collected cortical tissue.
Note: Make sure tissue is settled at the bottom of the conical tube prior to beginning aspiration.
11. Add 4 mL HBSS, 500 mL 2.5% trypsin, and 70 mL DNase to the conical tube with the cortical tissue and invert to mix well ( Figure 2A). a. Incubate in 5% CO2 incubator at 37 C for 15 min, inverting every 5 min to mix the cortical tissue and the HBSS/Trypsin/DNase solution.
CRITICAL: Monitor incubation at this step closely. It is important to not over incubate the cortical tissue with trypsin.
12. After the final inversion, wait for the cortical tissue to settle to the bottom of the conical tube then aspirate the HBSS/Trypsin/DNase solution. 13. Wash the cortical tissue twice with 10 mL HBSS, inverting each time to wash completely.
Note: Make sure tissue is settled at the bottom of the conical tube after inverting and prior to beginning each aspiration.
14. Add 2 mL HBSS and 30 mL DNase to the conical tube with the cortical tissue. With a P1000 pipette, homogenize the cortical tissue by slowly pipetting up and down 10 times ( Figure 2B). a. Homogenize the cortical tissue again with a P200 pipette, slowly pipetting up and down 10 times. 15. To a 50 mL conical tube, add 2 mL of 10% FBS/DMEM/PS media (warmed to 37 C). 16. Place a 70 mm cell strainer on top of the 50 mL conical tube. Wet the strainer with 10% FBS/ DMEM/PS media to minimize cell loss or cell death upon first contact with the strainer surface. 17. Transfer homogenized cortical tissue using a P200 pipette.

Mixed glial culture maintenance
Timing: As long as appropriate for the experiment (typically DIV 14-21) Note: A visualization of the development of the mixed glial culture at different timepoints (DIV 1-14) can be found in Figure 3.
23. After at least 24 h (DIV 1), the media should be aspirated and replaced with 20 mL of new 10% FBS/DMEM/PS (warmed to 37 C) to remove any debris. 24. At DIV 4 or DIV 5, the media should be changed again to remove any additional debris. Preparing plate for microglia plating Timing: 10 min hands-on, 2 h total 25. Prepare a 12-well plate for the assay 30 min before beginning microglia shake-off.
a. To each well, add one 18 mm glass coverslip. b. Add 600 mL 13 poly-l-lysine (PLL), described below under materials and equipment, to each well. Agitate the plate to make sure the cover glass is completely submerged. 26. Incubate the 13 PLL-coated plate for at least 2 h (at 37 C).
a. Every hour, agitate the plate ensure cover glass is well coated. 27. Near the end of the microglia shake-off, aspirate the 13 PLL and wash the coated wells with cell culture grade water. Aspirate and repeat wash for a total of two washes with cell culture grade water. a. The plate is now ready to be plated with cell/media suspension.
Note: Wait to remove the 13 PLL from the 12-well plate until cell/media suspension is ready to be added. If necessary, leave 13 PLL in 12-well plate and incubate at 37 C longer than 3 h. However, it is not ideal to leave plate incubating for much longer, so timing is important.

Collection and seeding of microglia
Timing: 1 h hands-on, 25 h total 28. Cover the T-75 flasks containing mixed glia culture to be shaken in aluminum foil. Place the covered flasks on shaker and securely attach the flasks with tape ( Figure 2C). 29. Shake the T-75 flasks at 230 rpm at 18 C-26 C for 3 h -3 h 30 min. Check after 3 h shaking under the microscope and decide whether they will need longer shaking. 30. Once the shake-off is complete, immediately collect the media containing microglia from the flasks with a pipette. a. Shaken-off cells should be collected into a 50 mL conical tube.
Note: Shaking-off microglia from the non-adherent floating cell layer is a commonly used method to isolate microglia from primary mixed glial cultures. 6,7 This method yields high-purity microglia cultures, with an expected purity of > 90%. 8 CRITICAL: After shake-off, the isolated microglia must be immediately plated. If the cells/ media remain still (either in a T-75 flask or conical tube), the microglia are likely to reattach to the bottom of the container.  CRITICAL: Prior to beginning an experiment and treating the newly plated microglia, cells should be checked for health and attachment. Any discrepancies between wells should be noted and taken into consideration when proceeding with the experiment. Examples of plated unhealthy and healthy cells are visualized in Figure 4. If wells have unhealthy cells, please consult troubleshooting problem 1.

LPS treatment to microglia
Timing: 30 min hands-on, up to 25 h total Please refer below to Table 1 for LPS stock dilution and Table 2 for an example of LPS treatment preparation.
Note: For LPS treatment and during the rest of the experiment, 1% FBS/DMEM/PS is used as a higher percentage of FBS can interfere with experimental conditions. Warm 1% FBS/DMEM/ PS at 37 C before adding to wells.
Note: As a control for LPS treatment, we recommend using the same volume of 13 dPBS.
37. Using the 1 mg/mL LPS Stock (see materials and equipment), prepare LPS treatments according to Tables 1 and 2.
Note: Revise the components of Table 2 according to desired conditions for individual experimental set-up.
38. Aspirate the media from the 12-well plate, making sure not to disturb the seeded cells. Wash the seeded cells with 1 mL 1% FBS/DMEM/PS media (warmed to 37 C). 39. Immediately prior to treating seeded cells, aspirate the remaining media and add 800 mL of the LPS or dPBS treatment(s) prepared in media (based on Table 2). Note: An example LPS treatment (dose and length of time) strategy is outlined in Figure 5.
40. Incubate treated cells at 37 C for the desired length of time (suggested: 6, 12 and/or 24 h, see Figure 5).

Phagocytosis assay in microglia
Timing: 30 min hands-on, 2 h 30 min total Note: Before performing the phagocytosis assay, microglia seeded in a 12-well plate can be treated with LPS at the dosage and for the length of time (6, 12, and/or 24 h) of interest. An alternate form of microglia activation can be used such as IFNg+TNFa. 9 These pro-inflammatory stimulants result in microglia transitioning from basal state in which microglia surveil the environment to an activated pro-inflammatory state. This activated state will result in increased phagocytotic activity in microglia. If LPS stimulation fails to activate microglia, refer to troubleshooting problem 5.
41. Prepare 10 mL per well of bead treatment solution by mixing latex bead stock in a 1:10 ratio with 1% FBS/DMEM/PS. a. For example, for 12 wells, prepare 120 mL by adding 12 mL latex bead stock to 108 mL 1% FBS/ DMEM/PS. b. This prepared volume accounts for extra treatment solution as only 8 mL of the solution well be added per well. 42. Add 8 mL of bead mixture directly to treatment media already in wells, tilting plate to distribute evenly within the well.
Note: For appropriate treatment controls, incubate one well with 1% FBS/DMEM/PS prepared without fluorescent beads.
43. Incubate plates at 37 C for 2 h then aspirate beads and media without disturbing cells/cover glass. 44. Rinse wells with 1 mL 13 dPBS. Aspirate and repeat for two total washes with 13 dPBS. 45. Aspirate 13 dPBS from wells and add 1 mL 4% paraformaldehyde (PFA) (prepare 4% PFA as described above in materials and equipment). 46. Incubate wells with 4% PFA for 10 min at 37 C to fix cells. 47. Wash wells twice with 1 mL 13 dPBS. 48. Aspirate 13 dPBS from wells and add 1 mL 13 dPBS to each well for storage. 49. Protect plates from light by wrapping plates in aluminum foil and store at 4 C.
a. Plates can be stored at 4 C for up to 1 month before visualization and analysis.   Figure 6A). a. For example, for 12 wells, prepare 840 mL by adding 1.68 mL Anti-Iba1, Rabbit to 840 mL of blocking solution. b. This prepared volume accounts for extra 1 ab solution as only 60 mL of the solution will be added per coverslip. c. This 1 ab solution needs to be placed onto parafilm stretched over a 12-well plate in 60 mL ''bubbles'' for each coverslip.

Fluorescent bead treatment to microglia for phagocytosis
Note: Iba1 is a microglia-and macrophage-specific protein marker commonly used when immunostaining microglia. However, Iba1 alone does not confirm the present of microglia and exclude a potential macrophage contamination. Therefore, other antibodies can be used in addition to anti-Iba1 to confirm microglia and should be selected based on the investigator's need.
Antibody for P2RY12, a receptor downregulated during inflammatory response, is specific to identify non-activated microglia 10 although due to the LPS-induced activation of microglia suggested in this protocol, P2RY12 might not be an ideal target for identifying activated microglia without an additional primary antibody. Anti-transmembrane protein 119 (TMEM119) antibody is commonly used to distinguish microglia from peripheral macrophages, which would provide information on a potential macrophage contamination. 11 53. Each coverslip needs to be removed from its well using forceps and placed cell-side down into ''bubble'' on the parafilm. Carefully place the lid of the 12-well plate over the coverslips. 54. With forceps, carefully pick up the coverslips and place each cell-side down into the ''bubble'' on the parafilm. Cover with 12-well plate lid. 55. Incubate in the 1 ab solution for 8-18 h at 4 C, covered to protect from light.
Note: To differentiate background signal from the specific Anti-Iba1 signal, a negative control can be used. Prepare the negative control by adding Rabbit IgG Isotype control (5 mg/mL,  61. Return coverslips to the wells of a 12-well plate. Wash coverslips with 1 mL 13 PBS per well and incubate for 5 min. Repeat for a total 3 washes. 62. Using forceps, remove each coverslip from the wells and mount on microscope slides with 1-2 drops of Invitrogenä ProLongä Gold Antifade Mountant with DAPI (DAPI visualizes nuclear DNA. DAPI staining is used as a nuclei marker that can identify cells). a. Mount 2-3 coverslips on each slide. 63. Allow slides to dry at 18 C-26 C for 1-2 h, covered. 64. Coat the edges of each coverslip on the slide with a thin layer of clear nail polish. This seals the cover glass to the slides. 65. Allow nail polish to dry at 18 C-26 C for 8-18 h, covered. Store slides at 4 C for long term storage.
Note: Samples can be visualized on a Zeiss Confocal microscope or Zeiss fluorescence microscope with Apotome using Zeiss ZEN software (Figures 6B and 6C). Image analysis and phagocytosis quantification can be completed using Fiji -ImageJ software. See imaging and quantification below for additional information.
Note: While fluorescent imaging is used in this protocol, an additional method for bead quantification in cells is flow cytometry, which provides the benefit of rapid analysis with a higher throughput. Our protocol uses immunostaining, fluorescent imaging, and manual counting to analyze the number of phagocytosed beads per cell and visualize the morphology of microglia.

Imaging and quantification of phagocytosed fluorescent beads in microglia
Timing: 2 h hands-on, 2 h total (depending on the sample size, it can be shorter or longer) 66. Image microglia and beads using microscope; a Zeiss Confocal microscope or a Zeiss fluorescence microscope with Apotome and a 203 lens. 67. Prepare for imaging by determining an exposure time for each channel to be used; DAPI, fluorescent beads, and microglia.
Note: This exposure time needs to remain constant between imaging different slides in the experiment.
68. Determine the Z-stack number and Z-stack interval that will be used for imaging.
Note: Since microglia are typically about 10-15 mm thick and the diameter of the beads have a 0.5 mm mean particle size, we recommend the Z-stack interval to be between 0.5-1 mm to easily differentiate between beads. We recommend 10-15 Z-stacks depending on the chosen interval.
69. To begin imaging, find an area of the coverslip that includes multiple cells. Set up the first Z-position and last Z-position by focusing up and down using imaging software. Collect 2-3 Z-stack images per coverslip.
Note: Once image parameters have been optimized, all cells need to be imaged using the optimized exposure time, Z-stack size and Z-stack interval so that cells in the same experiment can be compared.
Note: For each genotype, treatment, and treatment length, 2-3 coverslips are plated, treated, assayed and immunostained for analysis. Following immunostaining, each coverslip is imaged and 2-3 images are collected for quantification per coverslip.

OPEN ACCESS
70. Following imaging, count beads per microglia using Fiji -ImageJ software. a. Within each image, assess every microglia (Iba1/DAPI positive) for co-localization of beads (i.e., 0+ beads) by viewing the images in each Z-stack. b. The Z-stack images provide information on bead size and shape so that individual beads can be identified within range of the microglia marker. 71. All beads per microglia values are averaged for each treatment and treatment length then statistically compared between each genotype.
Note: If latex beads do not fluoresce, please see troubleshooting problem 3, and if immunostaining appears to be nonspecific, please see troubleshooting problem 4. Following analysis, if treatment does not increase phagocytosis, please see troubleshooting problem 5.

EXPECTED OUTCOMES
The first part of this protocol describes how to dissect and culture glial cells from PND 2 murine cortical tissue. Murine brain dissection at PND 2 can be challenging due to the brain size, however, practice helps with skill development. We recommend performing dissection and culture trial runs with practice mice before performing an experiment. These steps must be mastered to ensure ideal microglia isolation and growth. When cultured properly, this dissection is a consistent and reliable method to obtain viable primary microglia from PND 0 to PND 10 mice.
The second part of this protocol describes how to induce and visualize microglia phagocytosis activity accomplished by microglia isolation, LPS treatment, incubation with fluorescent beads, and immunostaining. When microglia are activated with LPS, it is expected that there will be phagocytosis of the fluorescent beads, with multiple beads often found in one cell. A lack of increased phagocytic response to LPS treatment is addressed in troubleshooting problem 5.
Using the experiment described here, Fmr1 KO primary microglia, following treatment with both 1 ng/mL LPS and 100 ng/mL LPS, had higher phagocytosis activity than WT microglia treated similarly (Parrott et al. 1 ).

LIMITATIONS
This protocol has been successfully used with mice PND 0 to PND 10. Cultures collected at later ages take longer to populate to a useable experimental quantity in vitro. Adult primary microglia isolation has been previously demonstrated, however, isolation tends to be more difficult than postnatal primary microglia isolation. 6 While there are many advantages using an in vitro approach to assess microglial phagocytic activity, there are limitations as well. An in vitro experiment such as this lacks the typical cell-to-cell interactions of microglia and other cells observed in vivo. Many pathways and cell interactions involved in phagocytosis are potentially disrupted by isolating microglia which may impact microglia activity. Additionally, while LPS is commonly used as a model for sepsis shock, LPS does not directly cross the blood brain barrier when administered peripherally, therefore; in vitro LPS treatment of microglia does not directly mirror typical in vivo inflammation. 12 Instead of using LPS, pro-inflammatory cytokines such as IFNg+TNFa can also be used as mentioned in troubleshooting problem 5. Additionally, fluorescent beads are not a physiological material, but can be used as an indicator of phagocytosis. With the limitations of in vitro experiments, it is necessary to limit extrapolations of results to in vivo microglia activity. 13

Potential solution
The CO 2 percentage levels in the incubator may not be at 5% as identified by the incubator's internal monitors. Recalibrate the incubator as needed to achieve a consistent environment of 37 C, 100% humidity, and 5% CO 2 /95% air by using a Bacharach CO 2 Gas Analyzer Kit (Grainger, Item # 6T151, UNSPSC #41113102, UNSPSC v841113102, Mfr. Model #10-5000). This gas analyzer will directly measure CO 2 levels in the incubator to confirm what is displayed by the incubator's internal monitors.
If the cell density is too low when initially cultured, cells may not grow by DIV 14. If the flask appears empty or less than 50% confluent by DIV 14, the cell culture preparation is not successful. To increase the number of cells isolated for the initial culture, either increase the number of pups dissected or use a smaller flask, such as a T-25 flask (25 cm 2 culture area). If 2 pups at PND 2 do not provide enough cells for a T-75 flask, increase the number of pups while maintaining the 1:1 gender ratio. At later PND ages, 2 pups should be sufficient for culturing in a T-75 flask.
If the mixed glial culture is failing to grow when using pups older than PND 2-3, the media used can be changed for culturing and plating. Instead of using 10% FBS/DMEM/PS media and 1% FBS/ DMEM/PS media (described above in materials and equipment), use 15% FBS/DMEM/F-12/PS media and 1% FBS/DMEM/F-12/PS. DMEM/F-12 media (ThermoFisher Scientific, Cat. No. 11320082) contains Ham's F-12 medium, a nutrient mixture that provides additional supplements to the cells. Additionally, increasing the FBS concentration provides a higher supplement concentration that better supports the metabolic requirements of glial cells.
The culture seeding density may be too low. To a 12-well plate, we suggest the optimal cell density for plating for a phagocytosis assay should be about 5 3 10 4 cell/well. This density is optimal for analysis purposes as well as cell viability. In our experience, the minimum viable cell density for plating is about 3 3 10 4 cell/well. However, if the cells are consistently failing to grow or are unhealthy when plating an optimal density of cells, consider allowing more DIV for cells to reach 70%-80% confluence before seeding.
Cells may be unhealthy when adding to the 12-well plate. Before shaking, cells should be examined underneath a microscope to ensure their health and density. An example of healthy cells ready for shaking are demonstrated in Figure 3 from DIV 10-14. Alternatively, cells may not be viable after shaking if shaken for longer than 3.5 h or if the temperature is too low. After shaking, it is expected that the cell density of microglia from 4 cultured brains resuspended in 2-3 mL of media will be a least 16 3 10 4 cells/mL. If this number is much lower, cells density may be too low for plating. In that case, the resuspended cells should not be used for the phagocytosis assay. A new culture must be made, or the T-75 flask should be given 1 week for microglia proliferate and re-shaken. After cells have been plated, cells should be examined underneath a microscope to ensure their health and density. Examples of healthy and unhealthy plated cells are demonstrated in Figure 4.

Problem 2
Microglia do not attach to the coverslip.

Potential solution
The 13 PLL coating may not have incubated for long enough or had not fully coated the cover glass.
It is important to agitate the cover glass coated with 13 PLL about once per hour to ensure the entire coverslip is submerged. If the coverslip is not completely submerged using 600 mL 13 PLL, add more 13 PLL (up to 1 mL/well total) to submerge the coverslip.

Problem 3
Latex beads do not fluorescence when imaged.

Potential solution
Proper storage for the latex beads is necessary to preserve the fluorescent signal. The latex bead solution should be stored in a dry, well-ventilated area and protected from light at all times. Additionally, the beads used in the experiment may be changed from fluorescent red to an alternate fluorescent color to suit the needs of the investigator.

Potential solution
Proper storage is necessary to maintain the reactivity of the antibody. Contact the antibody supplier to optimize storage conditions if needed. Depending on the needs of the investigator, the antibody targets may be adjusted and optimized as needed. However, these targets confirm cells (nuclei staining: DAPI) and identify cell type (microglia: Iba1).
Antibody incubation durations and concentrations used for immunostaining may need to be optimized when establishing this protocol within a new lab. The conditions and antibody concentrations used in this experiment can be optimized within the lab for the use of the specific antibodies. Additionally, the temperature at which the antibody is incubated can be adjusted. For example, instead of incubating the 1 antibody solution at 4 C for 8-18 h, try incubating it at 18 C-26 C for 4-6 h.

Problem 5
Microglia do not have increased phagocytosis activity in response to LPS treatment.

Potential solution
This problem is most likely due to low phagocytosis activity in microglia, due to the age and/or health of the cells. Previous work demonstrated that it is possible to successfully harvest microglia following repetitive shake-offs from a single culture, however, the magnitude of responsiveness to LPS treatment decreases with each shake-off. 3 To minimize this impact on experiments, only compared data collected from the same experiment and same shake-off. Additionally, try to use first or second shake-offs and limit the use of third and fourth shake-offs when possible.
Alternate forms of cell activation may be used to assess phagocytic activity. Other pro-inflammatory stimuli such as IFNg or TNFa cytokines have also been used to stimulate microglia cultured from rats, however, the cytokines were found to be less effective at creating a robust-inflammatory response in microglia. 9

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hye Young Lee (leeh6@uthscsa.edu).

Materials availability
The study did not generate new unique reagents.
Data and code availability All data reported in this paper will be shared by the lead contact upon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.