Prepare a solution of PBS, pH 7.2, 2mM EDTA and 0.5% BSA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution.
Notes:
Transfer mouse spleen into the gentleMACS C Tube containing the following amount of buffer:
1–2 mouse spleens: 3 mLNotes:
TexMACS™ medium supplemented with 2-Mercaptoethanol (final concentration 0.01 mM) and 100× penicillin/streptomycin stock solution (final concentration 1% ) and 50 U/mL Mouse IL-2.
Notes:
Notes:
Recommended procedure:
Make fresh T cell medium with TexMACS™ medium supplemented with 2-Mercaptoethanol (final concentration 0.01 mM) and 100× penicillin/streptomycin stock solution (final concentration 1% ) and 50 U/mL Mouse IL-2 (see "Staining for analysis of cell proliferation" for more details).
▲ Note: Make sure to freshly add IL-2 to the T cell medium for cell expansion.
Recommended reconstitution: 0.1mg/mL by reconstituting a 100 µg vial of mouse IL-2, research grade with 1 mL deionized sterile-filtered water. This results in a final activity of 500 U/µl.
Notes:
Prepare the following master mix fresh for each sample:
20 µL buffer, 5 µL of each antibody:
Store mastermix in the dark in the refrigerator (2−8 °C) until use. Do not store for extended periods.
Prepare the following master mix fresh for each sample:
15 µL buffer, 5 µL of each antibody:
Store mastermix in the dark in the refrigerator (2−8 °C) until use. Do not store for extended periods.
Notes:
Activation markers are typically analyzed on days 1 and 2 after activation with the T Cell Activation and Expansion Kit, mouse. However, please feel free to modify checkpoints according to your experimental needs.
Note:
For further flow cytometry analysis, it is recommended to remove the MACSiBead Particles from the cell suspension.
Make fresh T cell medium with TexMACS™ medium supplemented with 2-Mercaptoethanol (final concentration 0.01 mM) and 100× penicillin/streptomycin stock solution (final concentration 1% ). See "Staining for analysis of cell proliferation" for more details.
▲ Note: Restimulation of cells is performed in the absence of IL-2.
Notes:
Notes:
Notes:
Notes:
Notes:
Design your assay using two columns of the MACSPlex Filter Plate for the standards. Add each of the 7 standard samples in duplicates next to each other. Standards should be run in order from the lowest concentration (blank control: 0 pg/mL) to the highest concentration (stock solution: 10,000 pg/mL or 50,000 pg/mL). Start with the unknown sample in the next column of the plate. For details, see figure above.
Notes:
Notes:
The kit includes MACSPlex Setup Beads for flow cytometer instrument setup. MACSPlex Setup Beads are not required when using the MACSQuant Analyzer or MACSQuant Analyzer 10 but for all other instruments. The kits is not suitable for use with the MACSQuant VYB.
To perform the acquisition and data analysis of the MACSPlex Cytokine 10 Kit, mouse with the MACSQuant Instrument, it is recommended to use the Express Modes MACSPlex_Standard and MACSPlex_Sample to achieve automated measurement and data analysis. For details, refer to the relevant special protocol under the Library tab of the product page. The minimum version number of the Express Mode package needed to run the assay on the MACSQuant Instrument can be found there as well. To check the version number of the Express Mode package available on your MACSQuant Instrument, please select Help> Info> expressModes within the MACSQuantify Software. The version number of the Express Mode package increases with each Express Mode update. Make sure the MACSQuant Instrument contains an Express Mode package with at least the same or higher version number than the special protocol is marked with.
MACSiMAG™ Separator (#130-092-168)
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