Using Jurkat cells as an example, the cell labeling technique for the efferocytosis test is as follows [1]: 1. Apply 5 μg/mL staurosporine to RPMI-1640 culture media at 37°C and 5% CO2, then incubate the mixture for 3 hours at a density of 2.5 ×106 cells/mL to induce cell apoptosis. 2. To label cells, wash them in 1× DPBS and resuspend them in 2×107 cells/mL of Diluent C that contains either PKH26 (red fluorescence) or PKH67 (green fluorescence). 3. Rinse cells twice in 10% HI-FBS-containing DMEM basal medium. When doing exocytosis experiments, prepared cells ought to be employed right away. The following is how the product is used: 1. The PKH 67 working solution is prepared as follows: 1.1 Use 1 mL of preheated Diluent C to dilute 2 μL of the PKH 67 mother solution at a ratio of 1:500. Note: Please prepare PKH 67 working fluid for use by adjusting its concentration to suit the current circumstances. 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. The density of cells is 1×106/mL. 2.2 Incubate for 10 to 45 minutes at room temperature after adding 1 mL of PKH 67 working solution. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Adherent cell staining (3.1): Grow adherent cells on sterile coverslips. 3.2 Aspirate extra culture medium after removing the coverslip from the medium. 3.3 Add 100 μL of the dye working solution, give the cells a gentle shake to cover them completely, and then let them sit for 10 to 45 minutes. 3.4 Aspirate the dye working solution, wash twice, giving the culture medium five minutes between washes, and use a fluorescence microscope to observe. 4. Exosome labeling 4.1 Add 5–10 μL of the dye working solution to the exosomes (using, for example, 200 μg protein/150 μL PBS). 4.2 Give the exosome surface a gentle shake to ensure it is fully covered, then let it sit at room temperature in the dark for 30 to 60 minutes. 4.3 To prepare fluorescently labeled exosomes, excess dye must be removed by chromatography or ultrafiltration. Ultrafiltration: For centrifugal separation and purification, use a 100 kD ultrafiltration tube. Centrifugation is performed at 20–25°C, 7000–10,000 g, for 15 minutes, four times, and then the precipitate is collected. Spin column chromatography: Centrifugation conditions are 20–25 °C, centrifuge at 1000–2000 g for 1-3 minutes, and collect the precipitate. Column chromatography conditions include CORE400 packing and PBS buffer (pH 7.4) as the mobile phase. 4.4 Gather the exosome-labeled purified samples. 5. Observations 1. Kindly modify the PKH 67 working fluid concentration based on the current circumstances. 2. This product may not be used for clinical diagnosis or treatment, nor may it be included into food or medication. It is intended solely for professional use in scientific study. 3. Please wear disposable gloves and a lab coat for your health and safety.

[1]. Shi J, et al. A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis. Nat Commun. 2022 Dec 24;13(1):7929.
[2]. He L, et al. Intelligent manganese dioxide nanocomposites induce tumor immunogenic cell death and remould tumor microenvironment[J]. Chemical Engineering Journal, 2023: 141369.

257277-27-3

Typically exists as solids (or liquids in special cases) at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)