ER-Tracker dye is a derivative of BODIPY series dyes coupled with Glibenclamide, highly selective binding to the endoplasmic reticulum, non-toxic to cells at low concentrations, this type of dye is an environmentally sensitive probe, and formaldehyde treatment can still retain part of the fluorescence, with high fluorescence life, good extinction coefficient and other characteristics. Glibenclamide is an atp-dependent K + channel blocker (Kir6, KATP) and CFTR Cl-channel blocker that binds in the endoplasmic reticulum. ER-Tracker is not suitable for staining cells after fixation. Ex/Em=587/615 nm [1].
In Vitro, Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.) 1. Preparation of ER-Tracker Solution 1.1 Preparation of Storage Solution Prepare a 1 mM solution using anhydrous DMSO. 1.2 Preparation of working solution Dilute the stored solution with preheated serum-free cell culture medium or PBS to prepare an ER-Tracker working solution with concentrations ranging from 100 nM to 1 μM. Note: Please adjust the concentration of the ER-Tracker working solution according to the actual situation, and prepare it as needed. 2. Cell Staining (Suspension Cells) 2.1 Centrifuge to collect the cells, then add PBS for two washes, each for 5 minutes. The cell density is 1×106 per mL. 2.2 Add 1 mL of ER-Tracker working solution and incubate at room temperature for 5 to 30 minutes. 2.3 400 grams, centrifuge for 3-4 minutes, then discard the supernatant. 2.4 Add PBS to wash the cells twice, for 5 minutes each time. 2.5 After resuspending the cells in 1 mL of serum-free medium or PBS, observe them using a fluorescence microscope or a flow cytometer. 3. Cell Staining (Monolayer Cells) 3.1 Place the monolayer cells on a sterile cover slip. 3.2 Remove the cover glass from the culture medium and aspirate away the excess medium. 3.3 Add 100 μL of the dye working solution and gently shake to ensure complete coverage of the cells. Incubate for 5 to 30 minutes. 3.4 Remove the dye working solution and wash with the culture medium 2-3 times, each time for 5 minutes. Observe under a fluorescence microscope. Note: If flow cytometry is to be used for detection, the cells should first be digested with trypsin, resuspended, and then stained.
References:
[1]. Tanuja T Merianda, et al. A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins. Mol Cell Neurosci. 2009 Feb;40(2):128-42.
[2]. Merianda TT, et al. A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins. Mol Cell Neurosci. 2009 Feb;40(2):128-42.
[3]. Adiki Raja Sekhar, et al. A cell-permeant small molecule for the super-resolution imaging of the endoplasmic reticulum in live cells. Org Biomol Chem. 2019 Apr 10;17(15):3732-3736.
In Vitro, Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.) 1. Preparation of ER-Tracker Solution 1.1 Preparation of Storage Solution Prepare a 1 mM solution using anhydrous DMSO. 1.2 Preparation of working solution Dilute the stored solution with preheated serum-free cell culture medium or PBS to prepare an ER-Tracker working solution with concentrations ranging from 100 nM to 1 μM. Note: Please adjust the concentration of the ER-Tracker working solution according to the actual situation, and prepare it as needed. 2. Cell Staining (Suspension Cells) 2.1 Centrifuge to collect the cells, then add PBS for two washes, each for 5 minutes. The cell density is 1×106 per mL. 2.2 Add 1 mL of ER-Tracker working solution and incubate at room temperature for 5 to 30 minutes. 2.3 400 grams, centrifuge for 3-4 minutes, then discard the supernatant. 2.4 Add PBS to wash the cells twice, for 5 minutes each time. 2.5 After resuspending the cells in 1 mL of serum-free medium or PBS, observe them using a fluorescence microscope or a flow cytometer. 3. Cell Staining (Monolayer Cells) 3.1 Place the monolayer cells on a sterile cover slip. 3.2 Remove the cover glass from the culture medium and aspirate away the excess medium. 3.3 Add 100 μL of the dye working solution and gently shake to ensure complete coverage of the cells. Incubate for 5 to 30 minutes. 3.4 Remove the dye working solution and wash with the culture medium 2-3 times, each time for 5 minutes. Observe under a fluorescence microscope. Note: If flow cytometry is to be used for detection, the cells should first be digested with trypsin, resuspended, and then stained.
References:
[1]. Tanuja T Merianda, et al. A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins. Mol Cell Neurosci. 2009 Feb;40(2):128-42.
[2]. Merianda TT, et al. A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins. Mol Cell Neurosci. 2009 Feb;40(2):128-42.
[3]. Adiki Raja Sekhar, et al. A cell-permeant small molecule for the super-resolution imaging of the endoplasmic reticulum in live cells. Org Biomol Chem. 2019 Apr 10;17(15):3732-3736.