TITLE: Brain Homogenization and MSD Protocol for Mouse Brain and Serum
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details about the homogenization and MSD protocol of the mouse brain using various panel kits.

[STEPS]
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
1. Weight out brain using pre-chilled C-tube.
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
2. Use 2 mL RIPA (1x Halt PIC) per whole brain, 1 mL RIPA (1x Halt PIC) per half brain.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
3. Homogenize brain using “protein_01_01” routine on Miltenyi gentle MACS Octodissociator.
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
4. Vortex briefly and incubate 30 min on ice.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
5. Centrifuge at3500 rpm, 3 min, 4 °C.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
6. Transfer homogenate to two 1.5 mL eppendorf tubes.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
7. Vortex briefly and centrifuge at 14000 x g, 10 min, 4 °C.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
8. Transfer supernatant to new 1.5 mL eppendorf tubes

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
9. Perform 1:10 serial dilution. 
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
10. Blot 2 µL diluted sample onto Direct Detect assay card (run duplicates).

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
11. Use 2 µL RIPA (1x Halt PIC) as blank.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
12. Read assay card on Direct Detect Spectrometer.
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
13. Detectable range is 0.2 mg/mL to 5.0 mg/mL, dilute or concentrate as needed.

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
14. Average all readings for each sample to determine concentration.
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
3.1. Repeat homogenization routine if not fully homogenized.
SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
9.1. Dilute using 10 µL sample to 90 µL RIPA (1x Halt PIC) to achieve 1:100  dilution. (1/2)

SECTION: Brain Homogenization Using Miltenyi gentleMACS Octodissociator
9.2. Dilute using 10 µL sample to 90 µL RIPA (1x Halt PIC) to achieve 1:100  dilution. (2/2)
SECTION: Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
15. Using concentration determined, calculate volume needed for 200 µg protein.

SECTION: Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
16. Use RIPA(1x PIC) to dilute 200 µg protein to 100 µL (2 1799).
SECTION: Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
17. Combine 100 µL sample and 100 µL MSD diluent in U-bottom plate.

SECTION: Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
18. Load 50 µL diluted sample to MSD plate per duplicate.

SECTION: Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
19. Proceed with MSD assay protocol from manufacturer.
SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
20. Using concentration determined, calculate volume needed for 200 µgprotein.

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
21. Use RIPA(1x PIC) to dilute 200 µgprotein to 100 µL (2 1799).

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
22. Load 100 µL sample into U-bottom plate.

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
23. Add 20 µL 1 Molarity (M) HCl into U-bottom plate and shake for 10 min at 25 °C.

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
24. Neutralize sample with 14 µL 1.2 Molarity (M) NaOH in0.5 Molarity (M)HEPES.

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
25. Combine 100 µLtreated sample and 100 µL MSD diluent in new U-bottom plate.

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
26. Load 50 µLdiluted sample to MSD plate per duplicate.

SECTION: Brain MSD Protocol U-PLEX TGF-β Combo mouse
27. Proceed with MSD assay protocol from manufacturer.
SECTION: Serum MSD Protocol Proinflammatory Panel 1 mouse
28. Add 25 µL MSD Diluent to MSD Plate per duplicate.

SECTION: Serum MSD Protocol Proinflammatory Panel 1 mouse
29. Add 25 µL serum sample to MSD Plate per duplicate.

SECTION: Serum MSD Protocol Proinflammatory Panel 1 mouse
30. Proceed with MSD assay protocol from manufacturer.
SECTION: Serum MSD Protocol Cytokine Panel 1 mouse
31. Add 37.5 µL MSD Diluent to MSD Plate per duplicate.

SECTION: Serum MSD Protocol Cytokine Panel 1 mouse
32. Add 12.5 µL serum sample to MSD Plate per duplicate.

SECTION: Serum MSD Protocol Cytokine Panel 1 mouse
33. Proceed with MSD assay protocol from manufacturer.
SECTION: Serum MSD Protocol U-PLEX TGF-β Combo mouse
34. Add 50 µL serum sample to U-bottom plate.

SECTION: Serum MSD Protocol U-PLEX TGF-β Combo mouse
35. Add 10 µL 1 Molarity (M) HCl into U-bottom plate and shake for 10 min at 25 °C.

SECTION: Serum MSD Protocol U-PLEX TGF-β Combo mouse
36. Neutralize sample with7 µL 1.2 Molarity (M) NaOH in 0.5 Molarity (M) HEPES.

SECTION: Serum MSD Protocol U-PLEX TGF-β Combo mouse
37. Add 25 µL MSD Diluent to MSD Plate per duplicate.

SECTION: Serum MSD Protocol U-PLEX TGF-β Combo mouse
38. Add 25 µL treated serum sample to MSD Plate per duplicate.

SECTION: Serum MSD Protocol U-PLEX TGF-β Combo mouse
39. Proceed with MSD assay protocol from manufacturer.