VX-148

Collagen-induced arthritis (CIA) is an autoimmune murine model of rheumatoid arthritis in which immunization with type II collagen (CII) emulsified in an adjuvant induces a T cell- and autoantibody-driven inflammatory arthritis characterized by synovial hyperplasia, immune cell infiltration, and joint destruction. The model typically relies on genetically susceptible mouse strains (e. g. , DBA/1) and reproduces key features of human rheumatoid arthritis, including anti-collagen immune responses and progressive joint inflammation. Disease onset generally occurs within ~3-4 weeks after immunization, depending on antigen/adjuvant combinations and protocol variation. The immunopathology is driven by adaptive immune activation against CII, leading to systemic and local joint inflammation mediated by pro-inflammatory cytokines and effector immune cells, making CIA a standard preclinical platform for evaluating immunomodulatory and anti-arthritic interventions.

Imiquimod (IMQ)-induced psoriasiform dermatitis is a widely used murine model in which topical application of IMQ, a Toll-like receptor 7 (TLR7) agonist, triggers innate immune activation in the skin and induces a psoriasis-like inflammatory cascade characterized by epidermal hyperplasia, immune cell infiltration, and cytokine production dominated by the IL-23/IL-17 axis. This inflammatory response is mediated through activation of dendritic cells and downstream induction of IL-23, IL-17A, IL-22, and related pro-inflammatory mediators, recapitulating key features of human plaque psoriasis and enabling mechanistic and therapeutic studies. The model is commonly induced using Aldara (5% IMQ cream) applied topically to murine skin, resulting in rapid onset of erythema, scaling, and thickening that can be quantified as disease severity indices and validated histologically.

PBMC thawing for immune assays recovers viable cryopreserved peripheral blood mononuclear cells for downstream functional or phenotypic readouts, including ELISPOT, intracellular cytokine staining, proliferation assays, and flow-cytometric immunophenotyping. Cryopreserved PBMCs can support immune monitoring because antigen-specific T-cell function and major CD4/CD8 phenotypes may be retained after optimized freezing and thawing, although some lymphocyte subsets and activation or memory markers can be altered by cryopreservation. The technical objective is rapid warming of the frozen vial followed by controlled dilution and removal of DMSO-containing cryomedium, because thawing and wash conditions measurably affect viable PBMC recovery and downstream assay performance. Viability alone is insufficient for protocol evaluation because high viability may occur with low live-cell recovery, so both viable percentage and absolute live-cell recovery should be measured after thawing.

The following protocol uses the MTT colorimetric assay as a classic literature-established method for assessing cell viability/metabolic activity in cultured mammalian cells. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by metabolically active cells to a colored formazan product; the amount of formazan is quantified spectrophotometrically and provides an indirect measure of metabolically active viable cells. Importantly, MTT reduction reflects cellular oxidoreductase/metabolic activity rather than an absolute direct count of living cells, so changes in cellular metabolism can alter the signal independently of cell number.