siRNA against Sig1R was obtained from Life Technologies. A mitochondrial Case12‐expressing plasmid (pCase12‐mito) was purchased from Evrogen (Moscow, Russia). N2a‐IP3R3 cells were seeded at 2.0 × 105 cells/dish on poly‐D‐lysine‐coated glass‐bottom dishes (MatTek, Ashford, MA). After transfection with Lipofectamine 2000 and/or Lipofectamine RNAi MAX (Life Technologies), the cells were incubated for 24 h in the differentiation media. Fluo‐4 AM (Life Technologies), PRE‐084, or NE‐100 (both from Sigma‐Aldrich, 5 μM each) was added 1 h prior to observation. The cells were washed twice with warmed Hank's balanced saline (Life Technologies). Fluorescence intensity was measured every 3 s until 120 s, and intracellular Ca2+ release was stimulated by adding adenosine triphosphate (ATP; 10 μM) to the media at 30 s.
Pcase12 mito
Manufactured by Evrogen
PCase12-mito is a fluorescent protein engineered for mitochondrial targeting. It is designed to be expressed in the mitochondria of living cells, allowing for the visualization and study of mitochondrial structure and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pre 084, by Merck Group (1 mentions)
Ne 100, by Merck Group (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Poly d lysine coated glass bottom dishes, by MatTek (1 mentions)
Pdsred2 mito, by Takara Bio (1 mentions)
On target plus mouse sirnas, by Horizon Discovery (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Histamine, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
3 protocols using pcase12 mito
1
Monitoring Intracellular Ca2+ Dynamics in N2a-IP3R3 Cells
2
Optimized siRNA and Plasmid Transfection Protocol
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For siRNA experiments, A2/29 cells (0.3 × 105/ml) were plated onto 12-well plates. After 24 h, cells were transfected with ON-TARGETplus mouse siRNAs (100 nM; Dharmacon): scrambled (D-001810–10-10–05), Tspo (L-040291–02-0005), or Atg5 (L-064838–00-0010), each composed of 4 different siRNAs to avoid off-target effects. The siRNAs were transfected using Lipofectamine 2000 (Invitrogen, 11,668–019) according to the manufacturer’s instructions. After 4 h, the medium was changed and the cells were incubated with fresh medium for 44 h.
For plasmids transfection, A2/29 cells seeded on coverslips were transfected with 1.5 µg of plasmid using TurboFect Transfection Reagent (Thermo Fisher Scientific, R0534) according to the manufacturer’s protocol. The following plasmids were used: pmRFP-GFP-MAP1LC3B (a gift from Dr. Inhee Mook-Jung, Seoul National University), pmRFP-MAP1LC3B (Addgene, 21,075; Tamotsu Yoshimori’s lab), pEGFP-PRKN generated by subcloning of Myc-PRKN from pRK5-Myc-PRKN (Addgene, 17,612; Ted Dawson’s lab) into pEGFP-C1 (Clontech, 6084–1), pCase12-mito (Evrogen, FP995), and pDsRed2-mito (Clontech, 55,858).
For plasmids transfection, A2/29 cells seeded on coverslips were transfected with 1.5 µg of plasmid using TurboFect Transfection Reagent (Thermo Fisher Scientific, R0534) according to the manufacturer’s protocol. The following plasmids were used: pmRFP-GFP-MAP1LC3B (a gift from Dr. Inhee Mook-Jung, Seoul National University), pmRFP-MAP1LC3B (Addgene, 21,075; Tamotsu Yoshimori’s lab), pEGFP-PRKN generated by subcloning of Myc-PRKN from pRK5-Myc-PRKN (Addgene, 17,612; Ted Dawson’s lab) into pEGFP-C1 (Clontech, 6084–1), pCase12-mito (Evrogen, FP995), and pDsRed2-mito (Clontech, 55,858).
3
Histamine-Induced Mitochondrial Response in DU145 Cells
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DU145 cells expressing mock or COMP were transiently transfected with 5 μg pCase12-mito (Evrogen) using Lipofectamine 3000 (Invitrogen). After 24 hours, the cells were detached and washed in warm PBS supplemented with 10% HI-FBS. After 5 min stimulation with 0 or 100 μM histamine (Sigma-Aldrich), the cells were immediately analysed by flow cytometry.
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