TO-PRO 1 

TO-PRO 1 is a DNA-binding fluorescent dye for non-living cells (Ex/Em=515 nm/531 nm). TO-PRO 1 can intercalate into base pairs of double-stranded DNA and produce stronger fluorescence. TO-PRO 1 is suitable for necrotic cells or late apoptotic cells with damaged cell membranes, showing green fluorescence under fluorescence microscopy or flow cytometry. TO-PRO 1 can be used to distinguish live cells from dead cells and distinguish cell membrane integrity. TO-PRO 1 can be attached to the surface of Feraheme (FH) nanoparticles (NPs) to obtain fluorescent dye-functionalized NPs for drug delivery studies^[1][2][3].

TO-PRO 1 (100 nM; 15 min) can accurately distinguish necrotic cells (binding to both) from live cells (showing only red autofluorescence) in flow cytometry experiments of Jurkat and HT-29 cells when co-stained with Annexin V-Cy5.5^[1].
TO-PRO 1 (64.5 μg/mL; 15 min) colocalizes with DAPI in confocal microscopy experiments and binds to nuclear DNA specifically labeled with green fluorescence in Jurkat cells treated with Camptothecin (HY-16560)^[1].
TO-PRO 1 (100 nM; 15 min) reflects cell death more accurately than 5-CFDA-AM (HY-131131) in viability assays of algal cells such as Brachiomonas submarina and Tetraselmis suecica^[2].
TO-PRO 1 can be used to directly measure live and dead cells adhering to diatoms^[3]. TO-PRO-1 can only stain dead cells. When TO-PRO-1 was used to stain the marine diatom Nitzchia closterium, live and dead cells were identified as red and yellow, respectively, under a blue-excited epifluorescence microscope. Live cells appear red due to the autofluorescence of intracellular chlorophyll, while dead cells appear yellow due to the fluorescence of TO-PRO-1^[3].
TO-PRO 1 in vitro staining protocol and procedures^[1]:
1. Cell preparation
Resuspend cells (e.g., Jurkat, HT-29, or algae) in phosphate-buffered saline (PBS) at a density of 1×10⁵-1×10⁶ cells/mL.
Induce cell necrosis by treatment with Camptothecin (HY-16560) (5 μM，10 h), 5-FU (HY-90006)/Oxaliplatin (HY-17371), or gamma irradiation (lethal dose: 2400-4800 Gy).
2. Staining protocol
Dye preparation: Dilute a 1 mM DMSO stock solution of TO-PRO 1 to 100 nM (for mammalian cells) or 64.5 μg/mL (for algae cells) in PBS.
Sample incubation: Add 5 μL of dye to 30 μL of cell suspension, mix gently, and incubate in the dark at room temperature for 15 min.
Washing (for algal cells): Centrifuge at 2,500 g for 2 min, discard the supernatant, and resuspend in fresh PBS to reduce background fluorescence.
3. Detection methods
① Fluorescence microscopy: Detect the green fluorescence of TO-PRO 1 using an epifluorescence microscope with a blue filter (excitation: 400-550 nm, emission: 531 nm).
Count at least 300 cells, and it is necessary to distinguish between dead cells (bright green nuclei) and live cells (red chlorophyll autofluorescence in algae or no green fluorescence in mammalian cells).
② Flow cytometry: Use excitation light at 488 nm and detect emission light at 530-550 nm.
Gate cells based on forward/side scatter and fluorescence intensity to quantify necrotic cell population.
4. Control Setup
Positive Control: 100% viable cells (untreated, expected low TO-PRO 1 binding).
Negative Control: 100% non-viable cells (irradiated killed, expected high TO-PRO 1 binding).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

645.38

C₂₄H₂₉I₂N₃S

157199-59-2

Liquid

Brown to red

C[N+](CCC[N+]1=C2C=CC=CC2=C(/C=C3SC4=CC=CC=C4N\3C)C=C1)(C)C.[I-].[I-]

Room temperature in continental US; may vary elsewhere.

Solution, -20°C, protect from light, 2 years

• [1]. Hoonsung Cho, et al. Fluorochrome-functionalized nanoparticles for imaging DNA in biological systems. ACS Nano. 2013 Mar 26;7(3):2032-41.  [Content Brief]

  [2]. Gorokhova E, et al. A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy. J Microbiol Methods. 2012 Jun;89(3):216-21.  [Content Brief]

  [3]. Okochi M, et al. Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment. Applied microbiology and biotechnology 51 (1999): 364-369.