To establish the stable cell lines expressing the fluorescence specifically in mitochondria or ER, MDA-MB 435S cells were transfected with the pEYFP-Mito or pEYFP-ER vector (Clontech, Mountain, CA, USA). Stable cell lines expressing pEYFP-Mito or pEYFP-ER (YFP-Mito or YFP-ER) were selected with fresh medium containing 500 μg/ml G418 (Calbiochem). To quantitatively measure the dilation of mitochondria and the ER induced by DMC, we analyzed the average width of the vacuoles originated from mitochondria and the ER in YFP-Mito and YFP-ER cells using AxioVision Rel. 4.8 software (Zeiss). More than 200 clearly identifiable vacuoles derived from mitochondria in 50 YFP-Mito cells and >200 clearly identifiable vacuoles derived from the ER in 50 YFP-ER cells per experiment, randomly selected, were measured in three independent experiments.
Peyfp mito
Manufactured by Takara Bio
Sourced in United States
PEYFP-Mito is a fluorescent protein used for visualizing mitochondria in live cells. It is a variant of the yellow fluorescent protein (YFP) that is specifically targeted to the mitochondria, allowing researchers to observe the dynamics and distribution of this organelle within the cellular environment.
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Lab products found in correlation
Peyfp er vector, by Takara Bio (2 mentions)
Doxorubicin, by Merck Group (1 mentions)
Lps from escherichia coli 055 b5, by Merck Group (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
Mcherry gfp lc3, by Addgene (1 mentions)
Timed pregnant cd1 mice, by Charles River Laboratories (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
5 protocols using peyfp mito
1
Quantifying Mitochondrial and ER Dynamics
2
Quantifying ER and Mitochondrial Dilation
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To establish the stable cell lines expressing the fluorescence specifically in mitochondria or the ER, MDA-MB 435S cells were transfected with the pEYFP-Mito or pEYFP-ER vector (Clontech, Mountain View, CA, USA). Stable cell lines expressing pEYFP-Mito or pEYFP-ER (YFP-Mito or YFP-ER) were selected with fresh medium containing 500 μg ml−1 G418 (Calbiochem). To quantitatively measure the dilation of the ER and mitochondria induced by treatment with bortezomib and/or nutlin-3, we analyzed the average width of the vacuoles originated from mitochondria and the ER in YFP-Mito cells and YFP-ER cells using AxioVision Rel. 4.8 software (Zeiss). More than 200 clearly identifiable vacuoles derived from the ER in 50 YFP-ER cells and more than 200 clearly identifiable vacuoles derived from mitochondria in 50 YFP-Mito cells per experiment were randomly selected and measured in three independent experiments.
3
Mitochondrial and Endoplasmic Reticulum Markers in Drug Studies
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LPS from Escherichia coli 055:B5, Methyl-aminolevulinic acid (Me-ALA), Doxorubicin, N-acetyl-L-cysteine (NAC), and BAPTA-AM were from Sigma Aldrich. The plasmid pEYFP-Mito (mitochondrial marker) (27 (link)) was from Clontech. The plasmid pEYFP-C1-KDEL-GFP (28 (link)) (endoplasmic reticulum marker) was kindly provided by Dr. Sergio Grinstein (University of Toronto, Canada). The plasmid pCRT-EGFP (29 (link)) (Green fluorescent protein-tagged calreticulin) was kindly provided by Dra. Marta Hallak (CIQUIBIC, Argentina).
4
Fluorescent Protein Plasmids for Imaging
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Timed pregnant CD1 mice were obtained from Charles River (Wilmington, MA), and α-syn KO mice were obtained from the Jackson Laboratory (Bar Harbor, ME). GFP was ligated in-frame at the C-terminus of α-syn and cloned into the pcDNA3.1 vector to generate the α-syn–GFP plasmid. Other plasmid constructs include APP-YFP (Kaether et al., 2000 (link)), synaptophysin-GFP (Kaether et al., 2000 (link)), and pEYFP-Mito (Clontech, Mountain View, CA). The following plasmids (followed by plasmid number) were obtained from Addgene (Cambridge, MA): GFP-Rab7 (12605), mRFP-Rab7 (14436), GFP-LC3 (21073), TrkB-GFP (32500), RFP-Ub (11935), and mCherry-GFP-LC3 (22418).
5
Fluorescent Protein Fusion Constructs
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The plasmids for MyrPalm-mEGFP14 (link), ssRFP-KDEL15 (link), GalT-RFP17 (link), H2B-GFP18 (link), CD3δ-GFP19 (link), TOMM20-GFP20 (link), PARL-GFP20 (link), PINK1-mCherry20 (link), TAP1-GFP35 (link), TAP2-RFP35 (link), wtPrP-GFP22 (link) and SKL-RFP15 (link) have been described previously. For CD3δ-mCherry, GFP from CD3δ-GFP was replaced by mCherry. To create mCherry-CD3δ-linker-YFP, we used YFP-CD3δ19 (link) and replaced YFP with mCherry. The resulting construct was combined with CD3δ-CFP19 (link) to create mCherry-CD3δ-CFP. This construct was further modified by adding YFP to its carboxyl-terminus to get mCherry-CD3δ-linker-YFP with CFP as linker sequence. For H2B-mCherry, GFP from H2B-GFP was replaced by mCherry. For pmCherry-Mito and pGFP-Mito, EYFP from pEYFP-Mito (Clontech) was replaced by mCherry and EGFP, respectively. Brefeldin A was purchased from Sigma-Aldrich and used at 5 μg/ml.
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