Brain Homogenization and MSD Protocol for Mouse Brain and Serum
Scott Vermilyea
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Abstract
This protocol details about the homogenization and MSD protocol of the mouse brain using various panel kits.
Attachments
Steps
Brain Homogenization Using Miltenyi gentleMACS Octodissociator
Weight out brain using pre-chilled C-tube.
Use 2mL RIPA (1x Halt PIC) per whole brain, 1mL RIPA (1x Halt PIC) per half brain.
Homogenize brain using “protein_01_01” routine on Miltenyi gentle MACS Octodissociator.
Repeat homogenization routine if not fully homogenized.
Vortex briefly and incubate 0h 30m 0s On ice.
Centrifuge at3500rpm,4°C.
Transfer homogenate to two 1.5mL eppendorf tubes.
Vortex briefly and centrifuge at 14000x g,4°C.
Transfer supernatant to new 1.5mL eppendorf tubes
Perform 1:10 serial dilution.
Dilute using 10µL sample to 90µL RIPA (1x Halt PIC) to achieve 1:100 dilution. (1/2)
Dilute using 10µL sample to 90µL RIPA (1x Halt PIC) to achieve 1:100 dilution. (2/2)
Blot 2µL diluted sample onto Direct Detect assay card (run duplicates).
Use 2µL RIPA (1x Halt PIC) as blank.
Read assay card on Direct Detect Spectrometer.
Detectable range is 0.2mg/mL to 5.0mg/mL, dilute or concentrate as needed.
Average all readings for each sample to determine concentration.
Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
Using concentration determined, calculate volume needed for 200µg protein.
Use RIPA(1x PIC) to dilute 200µg protein to 100µL (2).
Combine 100µL sample and 100µL MSD diluent in U-bottom plate.
Load 50µL diluted sample to MSD plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
Brain MSD Protocol U-PLEX TGF-β Combo mouse
Using concentration determined, calculate volume needed for 200µgprotein.
Use RIPA(1x PIC) to dilute 200µgprotein to 100µL (2).
Load 100µL sample into U-bottom plate.
Add 20µL 1Molarity (M) HCl into U-bottom plate and shake for 0h 10m 0s at 25°C.
Neutralize sample with 14µL 1.2Molarity (M) NaOH in0.5Molarity (M)HEPES.
Combine 100µLtreated sample and 100µL MSD diluent in new U-bottom plate.
Load 50µLdiluted sample to MSD plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol Proinflammatory Panel 1 mouse
Add 25µL MSD Diluent to MSD Plate per duplicate.
Add 25µL serum sample to MSD Plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol Cytokine Panel 1 mouse
Add 37.5µL MSD Diluent to MSD Plate per duplicate.
Add 12.5µL serum sample to MSD Plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol U-PLEX TGF-β Combo mouse
Add 50µL serum sample to U-bottom plate.
Add 10µL 1Molarity (M) HCl into U-bottom plate and shake for 0h 10m 0s at 25°C.
Neutralize sample with7µL 1.2Molarity (M) NaOH in 0.5Molarity (M) HEPES.
Add 25µL MSD Diluent to MSD Plate per duplicate.
Add 25µL treated serum sample to MSD Plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
