This application protocol describes a complete workflow for reliable and efficient mouse TH cell differentiation, starting with single-cell preparation, followed by isolation of naive CD4+ T cells and in vitro activation and differentiation, through to comprehensive cell analysis. It is demonstrated 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.
Prepare a solution of PBS, pH 7.2, 2 mM EDTA, and 0.5% BSA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution.
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 TH cell differentiation experiments, it is recommended to always add 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.
For T cell polarization, the cells should be resuspended in culture medium at 1×106 cells/mL. Plate the cells at a density of 1×106 cells/cm2. Both the dilution and the cell density are important to assure optimal stimulation and cell growth. The following table lists culture plate sizes suitable for different cell numbers. It also indicates the appropriate amount of medium to add.
Table 1: 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, Fixation/Permeabilization Solution 1 must be diluted 1:4 with 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).
To achieve the appropriate working concentration for safe permeabilization of cells, 10× Permeabilization Buffer must be diluted 1:10 with deionized or distilled water before use (i.e. 1 mL of 10× Permeabilization Buffer plus 9 mL of deionized/distilled water).
▲ Note: Before diluting, make sure that the buffer does not contain any precipitates.
▲ 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 plate.
MACSiMAG Separator (# 130-092-168)
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