Apr 05, 2024
Brain Homogenization and MSD Protocol for Mouse Brain and Serum
- Scott Vermilyea1
- 1University of Minnesota
Protocol Citation: Scott Vermilyea 2024. Brain Homogenization and MSD Protocol for Mouse Brain and Serum. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxprqkl8j/v1
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
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol details about the homogenization and MSD protocol of the mouse brain using various panel kits.
Attachments
320-681.pdf
54KB
Materials
Reagent, Kits and Equipment List
1.
Equipment
gentleMACS™ C Tubes
NAME
C Tubes
TYPE
gentleMACS™
BRAND
130-093-237
SKU
LINK
2. RIPA Lysis and Extraction BufferThermo FisherCatalog #89901
3. Halt PIC (ThermoFisher 1861279)
4.
Equipment
Direct Detect® Assay-free Cards
NAME
Assay-free Cards
TYPE
Direct Detect®
BRAND
DDAC00010-GR
SKU
LINK
Membrane cards used for protein quantitation in the Direct Detect® Infrared Spectrometer
SPECIFICATIONS
5. HCl (Millipore Sigma H1758-500ML)
6. NaOH (Millipore Sigma 415413-500ML)
7. HEPES Sigma AldrichCatalog #83264-500ML-F
8. Proinflammatory Panel 1 mouse MESO SCALE DIAGNOSTICS, LLC.Catalog #MSD K15048D
9. Cytokine Panel 1 Mouse KitMESO SCALE DIAGNOSTICS, LLC.Catalog #K15245D
10. U-PLEX TGF-β Combo mouse MESO SCALE DIAGNOSTICS, LLC.Catalog #MSD K15242K-2
11.
Equipment
Octo Dissociator with Heaters
NAME
Octo Dissociator
TYPE
gentleMACS™
BRAND
130-096-427
SKU
LINK
12.
Equipment
Direct Detect Spectrometer
NAME
Spectrometer
TYPE
Direct Detect®
BRAND
DDHW00010-WW
SKU
LINK
13. Meso Sector S 600 (MSD 1201)
Brain Homogenization Using Miltenyi gentleMACS Octodissociator
Brain Homogenization Using Miltenyi gentleMACS Octodissociator
43m
Weight out brain using pre-chilled C-tube.
Use 2 mL RIPA (1x Halt PIC) per whole brain, 1 mL 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 00:30:00 On ice .
30m
Centrifuge at3500 rpm, 4°C, 00:03:00 .
3m
Transfer homogenate to two 1.5 mL eppendorf tubes.
Vortex briefly and centrifuge at 14000 x g, 4°C, 00:10:00 .
10m
Transfer supernatant to new 1.5 mL eppendorf tubes
Perform 1:10 serial dilution.
Dilute using 10 µL sample to 90 µL RIPA (1x Halt PIC) to achieve 1:100 dilution. (1/2)
Dilute using 10 µL sample to 90 µL RIPA (1x Halt PIC) to achieve 1:100 dilution. (2/2)
Blot 2 µL diluted sample onto Direct Detect assay card (run duplicates).
Use 2 µL RIPA (1x Halt PIC) as blank.
Read assay card on Direct Detect Spectrometer.
Detectable range is 0.2 mg/mL to 5.0 mg/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
Brain MSD Protocol Proinflammatory Panel 1 mouse and Cytokine Panel 1 mouse
Using concentration determined, calculate volume needed for 200 µg protein.
Use RIPA(1x PIC) to dilute 200 µg protein to 100 µL (2 undetermined ).
Combine 100 µL sample and 100 µL MSD diluent in U-bottom plate.
Load 50 µL diluted sample to MSD plate per duplicate.
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
10m
Using concentration determined, calculate volume needed for 200 µg protein.
Use RIPA(1x PIC) to dilute 200 µg protein to 100 µL (2 undetermined ).
Load 100 µL sample into U-bottom plate.
Add 20 µL 1 Molarity (M) HCl into U-bottom plate and shake for 00:10:00 at 25 °C .
10m
Neutralize sample with 14 µL 1.2 Molarity (M) NaOH in0.5 Molarity (M) HEPES.
Combine 100 µL treated sample and 100 µL MSD diluent in new U-bottom plate.
Load 50 µL diluted sample to MSD plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol Proinflammatory Panel 1 mouse
Serum MSD Protocol Proinflammatory Panel 1 mouse
Add 25 µL MSD Diluent to MSD Plate per duplicate.
Add 25 µL serum sample to MSD Plate per duplicate.
Proceed with MSD assay protocol from manufacturer.
Serum MSD Protocol Cytokine Panel 1 mouse
Serum MSD Protocol Cytokine Panel 1 mouse
Add 37.5 µL MSD Diluent to MSD Plate per duplicate.
Add 12.5 µL serum sample to MSD Plate per duplicate.
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
10m
Add 50 µL serum sample to U-bottom plate.
Add 10 µL 1 Molarity (M) HCl into U-bottom plate and shake for 00:10:00 at 25 °C .
10m
Neutralize sample with7 µL 1.2 Molarity (M) NaOH in 0.5 Molarity (M) HEPES.
Add 25 µL MSD Diluent to MSD Plate per duplicate.
Add 25 µL treated serum sample to MSD Plate per duplicate.
Proceed with MSD assay protocol from manufacturer.