Henna dyes are widely used for labeling cells, organelles, toners, viruses and lipoproteins. Long-chain henna cyanines include DiO (DiOC18 (3)), DiI (DiIC18 (3)), DiD (DiIC18 (5) )) and DiR, as well as the dialkylphoenix dye DiA (4-Di-16-ASP) Used to mark membranes and other focal structures. DiIC16 (3) has a shorter alkyl substituent (C16) than DiI (C18). They have extremely high extinction factors, environment-dependent fluorophores, and short excited-state depletion in pyramid environments. They are oil-forming and weakly fluorescent in water, but are highly fluorescent and fairly photostable when incorporated into membranes or bound to lipophilic biomolecules. These optical properties make them ideal for staining cell plasma membranes. Once cells are identified, these dyes diffuse laterally within the plasma membrane, resulting in staining of the entire cell [ 1 DiO, DiI, DiD, and DiR exhibit distinct green, orange, red, and red fluorescence, respectively, thereby facilitating multicolor imaging of live cells and Flow cytometry analysis. DiO and DiI can be used with standard FITC and TRITC filters respectively. Among them, DiI and its analogs are the most commonly used because they usually exhibit very low cytotoxicity. In addition, DiI is widely used to measure lipoproteins such as LDL and HDL. The lipophilic radical dye DiA is also commonly used to determine metatracing [2]. General protocol 1. Prepare 2 staining solutions 1. Prepare stock solutions in DMF, DMSO or ethanol: Stock solutions should be 1-5 mM in dimethylformamide (DMF), dimethyl sub-solution (DMSO or ethanol DMSO). As a solvent for Di, DMF is equivalent to ethanol. The stock solution should be aliquoted and any unused solution needed and refrozen to below -20 °C. Avoid repeated freeze/thaw cycles. The solution can be stored for 6 b. Prepare working solution: Add the stock solution to a suitable buffer such as serum-free medium, HBSS or PBS to prepare a working solution of 1 to 5 μM. We do not recommend working on the storage cabinet for more than one day. 2. Resuspend cells at final concentration a. Centrifuge at 1000g for 3-5 minutes at 4°C, discard the supernatant, and wash twice with PBS for 5 minutes each time. The cell density is 1×106/mL. b. Add 1 mL of Di working solution, then c. Centrifuge at 4°C and 400g for 3-4 minutes and discard the supernatant. d. Wash twice with PBS for 5 minutes each time. e. Resuspend cells in serum-free cell culture medium or PBS, fluorescence 3. Adherent cells a. Culture adherent cells on sterile coverslips. b. Remove the coverslip from the culture dish and aspirate excess cells. c. Add 100 μL working liquid, shake gently to completely cover the cells, and keep for 5-30 minutes. d. Wash twice with culture medium for 5 minutes each time. Observe by fluorescence microscopy or flow cytometry.

DiI-labeled motor neurons can survive up to 4 weeks in culture and up to 1 year in vivo [1].

[1]. Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations. Neuron. 2000 Aug;27(2):219-25.

[2]. Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. J Comp Neurol. 1990 Dec 22;302(4):729-38.

[3]. Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. J Comp Neurol. 1990 Dec 22;302(4):729-38.

C59H97CLN2O4

933.89

932.714

41085-99-8

2762626

Solid powder

68ºC (dec.)(lit.)

19.061

0

5

36

66

1210

0

JVXZRNYCRFIEGV-UHFFFAOYSA-M

InChI=1S/C59H97N2.ClHO4/c1-7-9-11-13-15-17-19-21-23-25-27-29-31-33-35-41-50-60-54-46-39-37-44-52(54)58(3,4)56(60)48-43-49-57-59(5,6)53-45-38-40-47-55(53)61(57)51-42-36-34-32-30-28-26-24-22-20-18-16-14-12-10-8-2;2-1(3,4)5/h37-40,43-49H,7-36,41-42,50-51H2,1-6H3;(H,2,3,4,5)/q+1;/p-1

2-[3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole;perchlorate

DiI stain; DiIC18(3); DiI

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~12.5 mg/mL (~13.39 mM)

Solubility in Formulation 1: 1.25 mg/mL (1.34 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.25 mg/mL (1.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)