License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Protocol status: Working We use this protocol and it's working
Created: November 12, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 55080
Keywords: Homogenization, MSD Protocol, Mouse Brain and Serum, ASAPCRN
Brain Homogenization Using Miltenyi gentleMACS Octodissociator
Brain Homogenization Using Miltenyi gentleMACS Octodissociator
1 Weight out brain using pre-chilled C-tube.
2 Use Amount 2 mL RIPA (1x Halt PIC) per whole brain, Amount 1 mL RIPA (1x Halt PIC) per half brain.
3 Homogenize brain using “protein_01_01” routine on Miltenyi gentle MACS Octodissociator.
3.1 Repeat homogenization routine if not fully homogenized.
4 Vortex briefly and incubate Duration 00:30:00 Temperature On ice .
5 Centrifuge at Centrifigation 3500 rpm , 4°C , 00:03:00 .
6 Transfer homogenate to two Amount 1.5 mL eppendorf tubes.
7 Vortex briefly and centrifuge at Centrifigation 14000 x g , 4°C , 00:10:00 .
8 Transfer supernatant to new Amount 1.5 mL eppendorf tubes
9 Perform 1:10 serial dilution.
9.1 Dilute using Amount 10 µL sample to Amount 90 µL RIPA (1x Halt PIC) to achieve 1:100 dilution. (1/2)
9.2 Dilute using Amount 10 µL sample to Amount 90 µL RIPA (1x Halt PIC) to achieve 1:100 dilution. (2/2)
10 Blot Amount 2 µL diluted sample onto Direct Detect assay card (run duplicates).
11 Use Amount 2 µL RIPA (1x Halt PIC) as blank.
12 Read assay card on Direct Detect Spectrometer.
13 Detectable range is Amount 0.2 mg/mL to Amount 5.0 mg/mL , dilute or concentrate as needed.
14 Average all readings for each sample to determine concentration.
Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
15 Using concentration determined, calculate volume needed for Amount 200 µg protein.
16 Use RIPA(1x PIC) to dilute Amount 200 µg protein to Amount 100 µL ( Amount 2 Undetermined ).
17 Combine Amount 100 µL sample and Amount 100 µL MSD diluent in U-bottom plate.
18 Load Amount 50 µL diluted sample to MSD plate per duplicate.
19 Proceed with MSD assay protocol from manufacturer.
Brain MSD Protocol U-PLEX TGF-β Combo mouse
Brain MSD Protocol U-PLEX TGF-β Combo mouse
20 Using concentration determined, calculate volume needed for Amount 200 µg protein.
21 Use RIPA(1x PIC) to dilute Amount 200 µg protein to Amount 100 µL ( Amount 2 Undetermined ).
22 Load Amount 100 µL sample into U-bottom plate.
23 Add Amount 20 µL Concentration 1 Molarity (M) HCl into U-bottom plate and shake for Duration 00:10:00 at Temperature 25 °C .
24 Neutralize sample with Amount 14 µL Concentration 1.2 Molarity (M) NaOH in Concentration 0.5 Molarity (M) HEPES.
25 Combine Amount 100 µL treated sample and Amount 100 µL MSD diluent in new U-bottom plate.
26 Load Amount 50 µL diluted sample to MSD plate per duplicate.
27 Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol Proinflammatory Panel 1 mouse
Serum MSD Protocol Proinflammatory Panel 1 mouse
28 Add Amount 25 µL MSD Diluent to MSD Plate per duplicate.
29 Add Amount 25 µL serum sample to MSD Plate per duplicate.
30 Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol Cytokine Panel 1 mouse
Serum MSD Protocol Cytokine Panel 1 mouse
31 Add Amount 37.5 µL MSD Diluent to MSD Plate per duplicate.
32 Add Amount 12.5 µL serum sample to MSD Plate per duplicate.
33 Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol U-PLEX TGF-β Combo mouse
Serum MSD Protocol U-PLEX TGF-β Combo mouse
34 Add Amount 50 µL serum sample to U-bottom plate.
35 Add Amount 10 µL Concentration 1 Molarity (M) HCl into U-bottom plate and shake for Duration 00:10:00 at Temperature 25 °C .
36 Neutralize sample with Amount 7 µL Concentration 1.2 Molarity (M) NaOH in Concentration 0.5 Molarity (M) HEPES.
37 Add Amount 25 µL MSD Diluent to MSD Plate per duplicate.
38 Add Amount 25 µL treated serum sample to MSD Plate per duplicate.
39 Proceed with MSD assay protocol from manufacturer.