Determining Nanoparticle-Induced Toxicity in Neurons Using Microglia as a Surrogate Biosensor

Protocol

1. Microglial Cell Culture Maintenance

1. Warm cell culture medium (Dulbecco's Modified Eagle Medium; DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/neomycin (PSN) to 37 °C.
2. Obtain frozen stock of BV2 microglial cells at passage 18 - 25 from storage at -80 °C. Rapidly thaw cells in a 37 °C water bath.
3. Gently transfer cells to a 75 cm² vented flask containing 10 ml cell culture media.
4. Incubate flask in 5% CO₂ at 37 °C. Aspirate media after 24 hr and replace with fresh media. Grow cells until approximately 70 - 80% confluency.

2. Counting and Plating Cells

1. In a 37 °C water bath, warm DMEM and 1x-trypsin-Ethylenediaminetetraacetic acid (EDTA) solution.
2. Aspirate media from cells and add 500 µl of trypsin. Incubate at 37°C for 2 - 5 min.
3. Using a scraper, remove cells from the flask and suspend in 5 ml of DMEM. Pass cells through a 70 µm strainer into a 50 ml tube three times.
4. Count cells using a hemocytometer and seed 8,000 cells/well in a black-walled clear bottom plate in a final volume of 200 µl DMEM supplemented with 10% FBS and 1% PSN. Incubate plate in 5% CO₂ at 37 °C for 24 hr.
5. Remove old DMEM and replace it with 200 µl of warmed DMEM supplemented with 1% PSN to serum starve microglia. Incubate plate in 5% CO₂ at 37 °C for 24 hr.

3. Activating Microglia

1. In separate tubes, dilute silver nanoparticles (AgNPs) (0.01, 0.05 or 0.1 µg/ml) and vehicle/neutral control (sodium citrate, 0.04 mM) in serum-free DMEM supplemented with 1% PSN to final working concentrations.
2. Remove 100 µl of media from each well and replace with 100 µl of appropriate treatment compound.
3. Incubate plate in 5 % CO₂ at 37 °C for 24 hr.

4. Filtering Conditioned Media

1. Collect 200 µl of media supernatant from each well and transfer it into a filter (molecular weight cutoff of 10 kDa) with collection tube (1.5 to 2 ml capacity).
2. Centrifuge at 14,000 x g at room temperature for 15 min.
3. Discard the flow-through (supernatant with particles) and place the filter upside-down into a new collection tube.
4. Centrifuge at 1,000 x g at room temperature for 2 min.
   NOTE: The resulting filtered media (containing secreted cytokines) is concentrated six-fold.
5. Bring the volume of the concentrate to 400 µl with fresh DMEM supplemented with 10% FBS and 1% PSN and store on ice.

5. Hypothalamic Cell Culture Maintenance

1. Warm DMEM cell culture medium supplemented with 10% FBS and 1% PSN to 37 °C.
2. Obtain a frozen stock of hypothalamic (mHypo-A1/2) cells at passage 18 - 25 stored at 80 °C. Rapidly thaw the cells in a 37 °C water bath.
3. Gently transfer cells to a 75 cm² vented flask containing 10 ml cell culture media.
4. Incubate flask in 5% CO₂ at 37 °C. Aspirate media after 24 hr and replace with fresh media. Grow cells until they are approximately at 70 - 80% confluency.

6. Determining Hypothalamic Cell Toxicity

1. Warm DMEM and 1x-trypsin-EDTA solution in a 37 °C water bath.
2. Aspirate media from hypothalamic cells and add 500 µl of trypsin. Incubate at 37 °C for 2 - 5 min. Remove cells from the flask using a scraper, as described above in step 2.
3. Count cells using a hemocytometer and plate hypothalamic cells at 5,000 cells/well in a black-walled clear bottom plate in a final volume of 200 µl DMEM supplemented with 10% FBS and 1% PSN, and incubate overnight in 5% CO₂ at 37 °C.
4. Remove 100 µl of old media using a multichannel pipettor and add 100 µl of filtered concentrated conditioned media to a final concentration of 1x.
5. Incubate plate in 5% CO₂ at 37 °C for 24 hr.
6. Add 22 µl of resazurin reagent and incubate plate for 20 min in 5% CO₂ at 37 °C.
7. Using a multimode spectrophotometer, record fluorescence (560 nm EX/590 nm EM) to measure cell viability. Report results as relative fluorescence units (RFU).

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Materials

Name	Company	Catalog Number	Comments
Cells/Reagents
Mouse microglial cell line (BV2)	Interlab Cell Line Collection (Genoa, Italy)	ATL03001
Adult Mouse Hypothalamus Cell Line mHypoA-1/2	Cellutions Biosystems Inc.	CLU172
Dulbecco's Modified Eagle's Medium	Invitrogen	10313-039
Fetal bovine serum	PAA Labs	A15-751
Penicillin/Streptomycin/Neomycin	Thermo Fisher Scientific	15640-055
Trypsin-EDTA	Thermo Fisher Scientific	25200056
Silver nanoparticles (20nm)	Sigma-Aldrich	730793
PrestoBlue Cell Viability Reagent	Invitrogen	A13262
Mouse TNF-α ELISA Max Delux	Biolegend	430904
Lipopolysaccharide	Sigma-Aldrich	L4391
Sodium Citrate	Sigma-Aldrich	S4641
Equipment
96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile	Thermo Fisher Scientific	165305
Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane	EMD Millipore	UFC501008
SpectraMax M5 Multi-Mode Microplate	Molecular Devices	M5
Falcon 50mL Conical Centrifuge Tubes	Corning, Inc	14-432-22
Falcon Cell Strainers 70 μm	Corning, Inc	08-771-2
Tabletop centrifuge 5430	Eppendorf	22620560