This application protocol describes a complete workflow for reliable and efficient mouse TH cell differentiation, starting with single-cell preparation, followed by isolation of naïve CD4+ T cells and in vitro activation and differentiation, through to comprehensive cell analysis. We demonstrate that in vitro TH cell differentiation in the presence of TexMACS™ Medium leads to a higher expression level of the characteristic effector cytokines in the various TH subsets compared to RPMI 1640.
▲ Note: Depending on the cell number to be cultivated and differentiated per well, 6-, 12-, 24- or 48-well plates might be used. Please refer to the protocol to choose the appropriate cell culture dish.
MACSiMAG™ Separator (#130-092-168)
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.
Transfer mouse spleen into the gentleMACS C Tube containing the following amount of PBE buffer:1–2 mouse spleens: 3 mL
TexMACS™ medium supplemented with FBS (final concentration 10%), 2-mercaptoethanol (final concentration 0.01 mM) and 100× penicillin/streptomycin stock solution (final concentration 1%).
In order to obtain maximal reproducibility for your TH cell differentiation experiments, it is recommended to always dose recombinant cytokines at a defined unit dose in [U/mL]. To calculate the cell culture concentration in [ng/mL] corresponding to the concentration in [U/mL], apply the following formula:
Final culture concentration [ng/mL] = (60 U/mL)/(biological activity [U/mg]*) × (106)
* Refer to the corresponding data sheet or CoA to obtain the biological activity.
Plate sizes for in vitro T cell polarization.
|Total cell number||Medium volume to add||Culture plate||Well diameter|
|2.5×105||0.25 mL||96 well||0.64 cm|
|1×106||1.00 mL||48 well||1.13 cm|
|2×106||2.00 mL||24 well||1.60 cm|
|4×106||4.00 mL||12 well||2.26 cm|
|1×107||10.00 mL||6 well||3.50 cm|
Prepare fully supplemented T cell medium by adding cytokines and antibodies to the supplemented TexMACS Medium (see "Things to prepare in advance") as follows:
For TH1 cell polarization (CytoBox TH1, mouse):
For TH2 cell polarization (CytoBox TH2, mouse):
For TH17 cell polarization (CytoBox TH17, mouse):
▲ Note: Refer to "Things to prepare in advance" for conversion from U/mL to ng/mL.
For further flow cytometry analysis, it is recommended to remove the MACSiBead™ particles from the cell suspension.
To achieve the appropriate working concentration for safe fixation and permeabilization of cells, the Fixation/Permeabilization Solution 1 must be diluted 1:4 with the Fixation/Permeabilization Solution 2 (i.e., for 1×10⁶ cells use 0.25 mL of Fixation/Permeabilization Solution 1 plus 0.75 mL of Fixation/Permeabilization Solution 2).
▲ Note: Before dilution, make sure that the buffer does not contain any precipitates.
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