a-simplified-method-for-isolation-culture-retinal-pigment-epithelial

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Case Western Reserve University

CD40 expressed in Müller cells is a central driver of diabetic retinopathy. CD40 causes phospholipase Cγ1 (PLCγ1)-dependent ATP release in Müller cells followed by purinergic receptor (P2X)-dependent production of proinflammatory cytokines in myeloid cells. In the diabetic retina, CD40 and P2X upregulate a broad range of inflammatory molecules that promote development of diabetic retinopathy. The molecular event downstream of CD40 that activates the PLCγ1-ATP-P2X-proinflammatory cytokine cascade and promotes development of diabetic retinopathy is unknown. We hypothesise that disruption of the CD40-driven molecular events that trigger this cascade prevents/treats diabetic retinopathy in mice.

Disruption of retinal inflammation and the development of diabetic retinopathy in mice by a CD40-derived peptide or mutation of CD40 in Müller cells.

Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects mammals and birds throughout the world. This protocol describes murine models of acute T. gondii infection, toxoplasmic encephalitis and toxoplasma retinochoroiditis. T. gondii infection in severe combined immunodeficient (SCID) mice, deficient in T and B cells, has allowed for the study of T cell-independent mechanisms of defense against intracellular organisms, as described here. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii induce protection against challenge with virulent strains of the parasite. They have allowed studies of immunization and adoptive-transfer experiments. A protocol is provided for infection with these mutant strains. The EGS strain of T. gondii has the unique feature of spontaneously forming tissue cysts in cell culture. Dual fluorescent reporter stains of this strain have allowed the study of tachyzoite to bradyzoite transitions in vitro and in vivo. A protocol for in vitro and in vivo growth of this strain and tissue cyst isolation is provided. Genetic manipulation of T. gondii and mice has led to the development of parasites that express fluorescent proteins as well as mice with fluorescently labeled leukocytes. This together with the use of T. gondii that express model antigens and transgenic mice that express the appropriate T cell receptor have facilitated the in vivo study of parasite host-interaction. In addition, parasites that express bioluminescent markers have made it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite that includes these newer methodologies. In addition, support protocols address the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of acute T. gondii infection in mice Basic Protocol 2: Model of toxoplasmic encephalitis and toxoplasma retinochoroiditis in chronically infected mice Basic Protocol 3: Assessment of T. gondii invasion into neural tissue Basic Protocol 4: T. gondii infection in scid/scid (SCID) mice Basic Protocol 5: Infection with the uracil auxotroph strain CPS1-1 or the temperature-sensitive TS-4 strain of T. gondii Basic Protocol 6: In vivo and in vitro maintenance of the EGS strain of T. gondii Support Protocol 1: Assessment of progression of infection and immune response to T. gondii Support Protocol 2: Maintenance of a bank of T. gondii cysts of the ME49 strain Support Protocol 3: Maintenance of T. gondii tachyzoites using human foreskin fibroblasts Support Protocol 4: Maintenance of T. gondii tachyzoites in mice Support Protocol 5: Preparation of T. gondii lysate antigens Support Protocol 6: Isolation of T. gondii tissue cysts from brain.

Alyssa Hubal

Department of Pathology,
Division of Infectious Diseases and HIV Medicine,
Department of Medicine,
Division of Infectious Diseases and HIV Medicine, Department of Medicine

Case Western Reserve University

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Alyssa Hubal

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology,Division of Infectious Diseases and HIV Medicine,Department of Medicine,Division of Infectious Diseases and HIV Medicine, Department of Medicine

Case Western Reserve University

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Department of Pathology,
Division of Infectious Diseases and HIV Medicine,
Department of Medicine,
Division of Infectious Diseases and HIV Medicine, Department of Medicine