Endoplasmic Reticulum Probe Red

We recommend 100 µL of diluted 1X Endoplasmic Reticulum Probe Red per well when using a 96-well plate. This product provides enough probe for an entire 96-well plate when using 100 µL per well.

This probe employs a cell-permeant fluorescent dye that is selective for the endoplasmic reticulum in most cell types.

Components:
1 vial of lyophilized Endoplasmic Reticulum Probe Red
1 vial (100 μL) of DMSO

Storage:
Upon receipt of the kit, store all components at -20°C and protected from light.
Once the DMSO is added to the probe and reconstituted to make the 500X stock solution, store aliquots at -20°C and protected from light.
Avoid freeze thaw cycles.

Required Materials Not Included:
Stain Buffer: Phenol-free cell culture media (without serum), HBSS or PBS

Imaging Guidelines:
Ex/Em = 590/620 nm 
Fluorescence microscope filter set: Cyanine3/TRITC

Flow Guidelines:
Analysis in Cyanine3 or similar channel

Preparation of 500X Endoplasmic Reticulum Stock Solution:

1. Thaw the probe and DMSO at room temperature before starting the experiment.
2. To make the 500X Endoplasmic Reticulum Stock Solution, add 20 μL of DMSO to the vial of Endoplasmic Reticulum Probe and mix well.​
   Note: It is recommended to centrifuge the vial at >5000g for 5 minutes to pellet aggregates.
3. Store unused 500X Endoplasmic Reticulum Stock Solution at -20°C in single use aliquots to avoid freeze thaw cycles. Unused stock solution is stable for at least 2 weeks at ≤ -20°C.

Live-Cell Imaging Protocol:

1. Prepare the 1X Endoplasmic Reticulum Staining Solution by diluting the 500X Endoplasmic Reticulum Stock Solution 1:500 in phenol-free media, HBSS or PBS and mix well.
   Note: Desired final volume to be determined by user. We recommend 100 μL per well in a 96-well plate.
2. Remove media from live cells and add the desired volume of 1X Endoplasmic Reticulum Staining Solution.
3. Optional: Include live-cell nuclear counterstain such as DRAQ5™ (Cat. No. 424101).
4. Incubate cells at 37°C for 15 - 30 minutes.
5. Remove the 1X Endoplasmic Reticulum Staining Solution.
6. Gently wash cells twice with phenol-free media, HBSS or PBS.
7. Proceed immediately to live-cell imaging.

Flow Cytometry Protocol

1. Prepare cell suspension with 0.25 - 2.5 x10⁶ cells in 499 μL of phenol-free media, HBSS or PBS.
2. Add 1 μL of 500X Endoplasmic Reticulum Stock Solution to the cell suspension (1:500 dilution).
   Note: Staining conditions and probe concentration may have to be modified according to cell type and assay used.
3. Incubate cells at room temperature for 15 - 30 minutes.
4. Wash cells twice with PBS or equivalent buffer and then add desired volume of the same buffer.
5. Proceed to analysis using a Flow Cytometer with Cyanine3 or equivalent channel.

Endoplasmic Reticulum