Inflammasome Activation in Human RPE Cells

Primary human adult RPE (haRPE) cells were isolated from donor eyes and cultured as previously described [14 (link)]. RPE cells (250,000 cells/well, passage 2 or 3) were seeded into Primaria 6-well plates (Corning, Lowell, MA, USA) and allowed to grow for at least 1 month. For treatments, media was changed to 1% FBS. Cells were primed with 5 ng/mL Interleukin-1α (IL-1α) (R & D Systems, Inc., Minneapolis, MN, USA) and 500 ng/mL lipopolysaccharide (LPS) (Sigma, St. Louis, MO, USA) for 16 h. Cells were then treated with 5 µM rotenone (Sigma), 0.2 nM Bafilomycin A (Sigma), or 5 mM ATP (Sigma) to activate inflammasomes for 24 h.

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Ebeling M.C., Fisher C.R., Kapphahn R.J., Stahl M.R., Shen S., Qu J., Montezuma S.R, & Ferrington D.A. (2022). Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration. Cells, 11(13), 2075.

Publication 2022

Primaria 6-well plates Interleukin-1α (IL-1α) lipopolysaccharide (LPS) rotenone Bafilomycin A ATP

Adult Bafilomycin a Cells Donor Eyes Human Inflammasomes Interleukin 1α Interleukin 5 Lipopolysaccharide Rotenone

Corresponding Organization : Doheny Eye Institute

Other organizations : University at Buffalo, State University of New York

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Variable analysis

independent variables

Interleukin-1α (IL-1α) (5 ng/mL)
Lipopolysaccharide (LPS) (500 ng/mL)
Rotenone (5 µM)
Bafilomycin A (0.2 nM)
ATP (5 mM)

dependent variables

Not explicitly mentioned

control variables

Passage 2 or 3 RPE cells
Cell seeding density (250,000 cells/well)
Culture duration (at least 1 month)
Media conditions (1% FBS)

controls

Positive control: Cells primed with IL-1α and LPS
Negative control: Not explicitly mentioned

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