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Interferon regulatory factor 5 (IRF5) is a transcription factor that plays a central role in inflammatory responses. It mediates the induction of type I interferons (IFNs) and pro-inflammatory cytokines by binding to ISRE and NF-κB sequences, respectively. IRF5 is considered a key downstream factor of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). The mechanisms of IRF5 activation vary depending on the specific PRR being activated. TLR stimulation activates IRF5 downstream via the adaptor proteins MyD88 or TRIF. The sensing of nucleic acids by RIG-I, MDA5, cGAS, or STING activates IRF5 through TBK1. IRF5 can also be activated by the IKKβ kinase, acting downstream of the MAVS adaptor protein associated with RIG-I or MDA5. IRF5 plays a pivotal role in a variety of inflammatory and autoimmune diseases, including rheumatoid arthritis and inflammatory bowel disease.

The THP-1 Human IRF5-KO/NFκB-SEAP Cell model effectively simulates the in vivo TLR7/TLR8 signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the THP-1 Human IRF5-KO/NFκB-SEAP Cell model

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.