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1. Microglial Cell Culture Maintenance
- 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.
- 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.
- Gently transfer cells to a 75 cm2 vented flask containing 10 ml cell culture media.
- 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
- In a 37 °C water bath, warm DMEM and 1x-trypsin-Ethylenediaminetetraacetic acid (EDTA) solution.
- Aspirate media from cells and add 500 µl of trypsin. Incubate at 37°C for 2 - 5 min.
- 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.
- 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.
- 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
- 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.
- Remove 100 µl of media from each well and replace with 100 µl of appropriate treatment compound.
- Incubate plate in 5 % CO2 at 37 °C for 24 hr.
4. Filtering Conditioned Media
- 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).
- Centrifuge at 14,000 x g at room temperature for 15 min.
- Discard the flow-through (supernatant with particles) and place the filter upside-down into a new collection tube.
- Centrifuge at 1,000 x g at room temperature for 2 min.
NOTE: The resulting filtered media (containing secreted cytokines) is concentrated six-fold.
- 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
- Warm DMEM cell culture medium supplemented with 10% FBS and 1% PSN to 37 °C.
- 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.
- Gently transfer cells to a 75 cm2 vented flask containing 10 ml cell culture media.
- 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
- Warm DMEM and 1x-trypsin-EDTA solution in a 37 °C water bath.
- 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.
- 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.
- 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.
- Incubate plate in 5% CO2 at 37 °C for 24 hr.
- Add 22 µl of resazurin reagent and incubate plate for 20 min in 5% CO2 at 37 °C.
- Using a multimode spectrophotometer, record fluorescence (560 nm EX/590 nm EM) to measure cell viability. Report results as relative fluorescence units (RFU).