Committed to providing comprehensive support for global drug development

FOXP3 (Forkhead Box, FOX) belongs to the forkhead/winged-helix family of transcription factors. As a core regulatory factor for regulatory T cells (Tregs), it plays a pivotal role in the maintenance of immune tolerance, the pathogenesis of autoimmune diseases, and tumor immune evasion. Members of this gene family all bind to specific DNA sites via their conserved forkhead domain, thereby participating in the regulation of target genes.

FOXP3 forms complexes with various proteins and is subject to regulation through a range of post-translational modifications (PTMs), including acetylation, phosphorylation, ubiquitination, and methylation. Consequently, these post-translational modifications can alter the stability of FOXP3 as well as its capacity to regulate gene expression, ultimately influencing the activity of regulatory T cells. FOXP3 likely contributes to the maintenance of Treg cell function primarily through the following three pathways: (1) FOXP3 can competitively bind to genes associated with cellular activation, thereby indirectly inhibiting the transcription of effector genes; (2) FOXP3 can directly repress the expression of effector genes (e.g., the IL-2 gene); and (3) acting as an adaptor protein, FOXP3 can recruit other effector molecules with inhibitory functions to bind to target genes.

The FOXP3 promoter-Luc Jurkat reporter cell line consists of Jurkat cells in which the expression of a Luciferase (Luc) reporter gene is driven and regulated by the FOXP3 promoter. PMA is an analog of DAG (diacylglycerol) that can freely traverse the cell membrane; it is utilized to assess CD3/CD28-mediated T-cell activation, as illustrated by the principle depicted in the figure below.

Figure 1. Schematic diagram of the Jurkat E6.1 Human FOXP3 promoter-Luc cell model.

Figure 2. Induction of FOXP3 Activity by PMA  with Ionomycin in FOXP3 promoter-Luc Jurkat .

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.