Advice (This is our suggested protocol, which should be adjusted to suit your particular circumstances as it simply offers guidance). 1. DNA and RNA can be stained differently in unfixed cells using the following steps [1]: (1) Set the flow cytometer to excite at 488 nm, use an emission filter, and differentiate between red fluorescent dichroic mirror luminescence (measured above 640 or 650 nm) and green fluorescence (measured at 515-545 nm). (2) Pour a 0.2 mL portion of the first cell suspension into a little glass or plastic tube, such as one with a capacity of 2 or 5 mL. Remain cool on ice. The 0.4 mL of ice-cold cell permeabilization solution should be added gradually. Place the cells on ice and wait 15 seconds. (4) Gently stir in 1.2 mL of the ice-cold alkali staining solution with acridine orange. Store cells in the dark on ice. (5) Use a flow cytometer to measure and record the cell fluorescence two to ten minutes after adding the acridine orange alkali staining solution. 2. Differential labeling of fixed cells [1]: (1a) Rinse cells once in ice-cold PBS and suspend at ~106 cells/mL for cells in suspension culture or blood samples. (1b) For cells adhered to tissue culture plates: Gather cells by trypsinization from dishes or flasks, mix them with cells floating in the culture medium (primarily detached mitotic cells and dead cells), and then wash the mixture once with medium containing serum to render the cells inactive. Trypsin. Approximately 106 cells/mL were suspended in ice-cold PBS. (1c) For cells separated from solid tumors, rinse the cells in ice-cold PBS at a density of around 106 cells/mL, removing any enzymes that were employed to dissociate the cells. (2) Fill a 15 mL conical glass tube with 10 mL of ice-cold 70% ethanol and add 1 mL of the cell suspension using a Pasteur pipette. Put cells on ice for at least two hours. (3) Centrifuge the tube for five minutes at 4°C and 300 × g. Eliminate all ethanol, give the cells a single rinse in ice-cold PBS, and then resuspend them in the same PBS at a density of less than 2 × 106 cells/mL. (4) Pipette 0.2 mL of the cell suspension (≤2 × 105 cells) and put it in a tiny tube (such as one with a capacity of 2 or 5 mL). Remain cool on ice. (5) Pour in 0.4 milliliters of the icy permeabilization solution. Place the cells on ice and wait 15 seconds. (6) Add 1.2 mL of the acridine orange alkali staining solution, which should be ice cold. Set up the cells on ice. (7) Use a flow cytometer to measure and record the cell fluorescence two to ten minutes after adding the acridine orange alkali staining solution.

[1]. Darzynkiewicz Z, et al. Differential staining of DNA and RNA. Curr Protoc Cytom. 2004 Nov;Chapter 7:Unit 7.3.
[2]. Mirrett S. Acridine orange stain. Infect Control. 1982 May-Jun;3(3):250-2.
[3]. Yektaeian N, et al. Lipophilic tracer Dil and fluorescence labeling of acridine orange used for Leishmania major tracing in the fibroblast cells. Heliyon. 2019 Dec 18;5(12):e03073.

C17H19N3

265.35

494-38-2

Acridine Orange hydrochloride;65-61-2;Acridine Orange zinc chloride salt;10127-02-3

CN(C)C1=CC2=NC3=C(C=CC(=C3)N(C)C)C=C2C=C1

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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