Protocol for LipoLIVE™ Deep Red Non-Toxic Dye

In Brief

LipoLIVE™ Deep Red Non-Toxic Dye (Catalog # 9042) is a ready-to-use fluorescent probe for live-cell imaging of lipid droplet and neutral lipids. It does not require cell fixation or affect cell health and maintains the physiological integrity of models, enabling free-wash monitoring of lipid metabolism.

Designed for real-time confocal microscopy, and high-content screening (HCS) workflows, it offers exceptional signal-to-noise ratios and photostability.

LipoLIVE™ is a trademark of Saguaro Biosciences.

1. Protocol Overview

2. Content and Storage

Table 1. LipoLIVE™ Deep Red Non-Toxic Dye Product Information.

Intended Use: For research use only. Not for use in diagnostics or therapeutic procedures.

3. General Guidelines

LipoLIVE™ Deep Red Non-Toxic Dye Dilution and Preparation

• Warm up the LipoLIVE™ Deep Red Non-Toxic Dye tube to room temperature before use to avoid condensation to form and water to get into the anhydrous dye solution.
• Gently spin the tube before use to collect any dye solution that may remain near the cap.
• Dilute LipoLIVE™ Deep Red Non-Toxic Dye 1,000-fold in preferred culture medium.
• Vortex thoroughly.

NOTE: We recommend 1X as a starting point for optimization. Higher or lower concentrations may be optimal for different imaging systems and cell models.

• Seed cells at desired density (typically to achieve 70-80% confluence) in cell culture medium containing LipoLIVE™ Deep Red Non-Toxic Dye in a black multi-well plate. Return to the incubator at 37°C, 5% CO₂ overnight.
• No washing step is required prior to imaging. Keep LipoLIVE™ Deep Red Non-Toxic Dye in solution throughout the assay.

Alternative Cell Culture Indications for LipoLIVE™ Deep Red Non-Toxic Dye

• While we recommend seeding cells in the presence of diluted LipoLIVE™ Deep Red Non-Toxic Dye, the dye can be added after cell seeding, before or following compound addition. Optimisation of seeding density and incubation prior to imaging are required. For reference, LipoLIVE™ Deep Red Non-Toxic Dye staining stabilizes after 4 hours in U2OS cells (at a concentration of 1X).
• A nuclear dye can be added to allow cell segmentation during data analysis, such as NucleoLIVE™ Non-Toxic Dye (Cat. No. 8935) or NucleoLIVE™ Blue Non-Toxic Dye (Cat. No. 9011). We recommend running a preliminary imaging test on cells treated with single dyes to validate the staining kinetics and absence of fluorescence bleed-through between the nuclear dye and LipoLIVE™ Deep Red channels on your system.

4. Technical Specifications & Instrument Compatibility

LipoLIVE™ Deep Red Non-Toxic Dye is a fluorescent dye compatible with GFP, Cy3 or FITC filters.

Instrument compatibility of LipoLIVE™ Deep Red Non-Toxic Dye

LipoLIVE™ Deep Red Non-Toxic Dye is compatible with other high-content imagers and confocal microscopes.

5. LipoLIVE™ Deep Red Non-Toxic Dye, Allowing for High Quality Staining of Lipid Droplets and Neutral Lipids without Compromising Cell Health

Figure 2. LipoLIVE™ rapidly stains lipid droplets in live cells. Composite images of MCF-7 and HepG2 cells stained with LipoLIVE™ Deep Red for 24h before overnight incubation with 100 µM Oleic Acid.

Figure 3. LipoLIVE™ preserves normal cell proliferation and viability, even in sensitive models. U2OS cells and HCT116 cells showed comparable viability to DMSO controls after 72-hour incubation with LipoLIVE™ Deep Red.

6. Recommended Positive Control Compounds for Lipid Studies*

  Table 2. Compounds and concentration for U2OS cells in 2D.

* Compounds provided as examples only. Validation required for each experimental protocol.

7. Example Protocol (for kinetic, 2D live-cell assay)

U2OS cells are treated with standard compounds for ER stress and apoptosis. MCF7 are cultured in DMEM/F-12 complemented with 10% FBS and 1% Penicillin/Streptomycin.

LipoLIVE™ Deep Red Non-Toxic Dye Dilution and Preparation (Day 0):

• Warm up the LipoLIVE™ Deep Red Non-Toxic Dye tubes to room temperature before use and gently spin to collect any dye solution that may remain near the cap.
• Optional: Dilute 10 μL NucleoLIVE™ Non-Toxic Dye in the same 10 mL culture medium (1000-fold).
• Vortex thoroughly.

Cell Culture Protocol with LipoLIVE™ Deep Red Non-toxic Dye (Day 0):

• Harvest and count MCF7 cells.
• Resuspend cells in prepared culture medium with LipoLIVE™ Deep Red Non-Toxic Dye (and NucleoLIVE™ Non-Toxic Dye, if applicable) at 80,000 cells/mL.
• Seed 96-well plate with 100 μL cell suspension per well to a final density of 8,000 cells per well.
• Incubate overnight at 37°C, 5% CO₂.

Standard Compound Preparation and Addition (Day 1):

• Prepare dose-response curves with 10x concentrations, maintaining constant vehicle (0.1% DMSO) solvent concentration.
• Prepare negative controls with 0.1% DMSO in complete media.
• Distribute 12.5 μL of test compounds or controls per well.

Imaging and Data Acquisition (Days 1-3):

• Image 96-well plate at 3h, 6h and 24h after addition of test compounds.

8. Frequently Asked Questions about LipoLIVE™ Deep Red Non-Toxic Dye

• What is LipoLIVE™ Deep Red Non-Toxic Dye used for?

  LipoLIVE™ Deep Red Non-Toxic Dye is used for the real-time visualization of lipid droplet and neutral lipids in living cells, without affecting cell viability.

• Does LipoLIVE™ Deep Red Non-Toxic Dye require cell fixation?

  No, LipoLIVE™ Deep Red Non-Toxic Dye does not require cell fixation.

• Is a wash step required before imaging?

  No washing step is required prior to imaging.

• Which imaging systems are compatible with LipoLIVE™ Deep Red Non-Toxic Dye?

  LipoLIVE™ Deep Red Non-Toxic Dye is compatible with high-content imagers and confocal microscopes.