Operating Instructions (The following is our recommended operating procedure; this procedure is for reference only, please modify it according to your specific needs.) 1. Preparation of Stock and Working Solutions a. Prepare 10 mM CDr17 stock solution (dissolved in anhydrous DMSO). b. Dilute the stock solution with preheated serum-free cell culture medium or PBS to prepare 1 μM CDr17 working solution. Note: Please adjust the concentration of Rhobo6 working solution according to your specific experimental requirements and prepare it immediately before use. 2. Cell Staining a. Culture cells on sterile coverslips. b. Remove the coverslips from the culture medium and aspirate excess medium. c. Add the working solution, gently agitate to ensure complete coverage of the cells, and incubate at room temperature for 30 minutes to 1 hour. d. Aspirate the dye working solution, then wash 2-3 times with culture medium (5 minutes each time). Observe using a fluorescence microscope or flow cytometer. Note: For flow cytometry analysis, cells must first be digested with trypsin, separated, and resuspended before staining. CDr17 (1 μM; 1 h) selectively stained M1 macrophages differentiated from RAW264.7 cells, with a staining intensity 5 times higher than that of M0 or M2 macrophages [1]. D-glucose competitively inhibited the uptake of CDr17 (1 μM; 30 min) by M1 macrophages differentiated from RAW264.7 cells in a dose-dependent manner, while L-glucose had no such effect [1]. The universal GLUT inhibitor cytochalasin B inhibited the uptake of CDr17 (1 μM) by M1 macrophages differentiated from RAW264.7 cells in a dose-dependent manner [1]. The uptake of CDr17 (1 μM; 30 min) by M1 macrophages differentiated from RAW264.7 cells was reduced by 80% in GLUT1 knockout cells, confirming that GLUT1 is a target transporter [1]. CDr17 (0.5 μM; 30 min) selectively labels M1 macrophages differentiated from THP-1 human cells[1]. CDr17 selectively labels primary mouse peritoneal M1 macrophages, but not primary M0 and M2 macrophages[1]. In HeLa cells, the staining intensity of CDr17 (30 min) is correlated with the expression level of GLUT1, with stronger staining in GLUT1-overexpressing cells and weaker staining in GLUT1-inhibited cells[1].

1. In vivo imaging a. Take 10 mM CDr17 (dissolved in DMSO) stock solution and dilute it with sterile PBS to 1 mM working solution. b. Filter the solution through a 0.2 μm filter membrane for sterilization. Use immediately or aliquot into single-use portions and store at -20 °C, avoiding repeated freeze-thaw cycles and exposure to sunlight. c. 15 minutes before imaging, inject CDr17 working solution at a concentration of 0.5–1 mM intravenously. Note: CDr17 kinetic studies should be performed for each animal model to determine the peak signal time. CDr17 (500 μM; intravenous injection; single dose) selectively displays M1 macrophages in LPS-induced acute paw inflammation in C57BL/6J mice [1]. CDr17 (1 mM, 100 μL; intravenous injection; single dose) selectively displays inflamed joint regions in CAIA-induced rheumatoid arthritis in BALB/c mice, with fluorescence intensity correlated with disease severity [1]. CDr17 (1 mM; intravenous injection; single dose) selectively displays inflammatory joint regions in CIA-induced rheumatoid arthritis in DBA/1J mice [1].

Animal/Disease Models:C57BL/6J (male, 6 to 8 weeks old) [1]
Doses: 500 μM
Route of Administration: Intravenous injection; single dose
Experimental Results: Compared with the control tissue injected with PBS, the number of stained cells in the LPS-injected inflamed claw tissue was significantly increased. Anti-CD86 co-staining confirmed that the stained cells were M1 macrophages.

---

Animal/Disease Models:BALB/c (female, 9 or 10 weeks old) [1]
Doses: 1 mM
Route of Administration: Intravenous injection; single dose
Experimental Results: Fluorescence intensity peaked 15 minutes after injection. Compared with control mice, the fluorescence intensity in the inflamed joint areas of RA mice was significantly higher. The degree of joint swelling was significantly positively correlated with CDr17 fluorescence intensity (R2 = 0.8220). This confirmed that CDr17 signaling was co-localized with CD68-positive macrophages in the synovium of RA mice.

---

Animal/Disease Models:DBA/1J (female, 9 weeks old) [1]
Doses: 1 mM
Route of Administration: Intravenous injection; single dose
Experimental Results: CIA-induced rheumatoid arthritis joints and normal control joints were successfully distinguished by selective fluorescence signals.

[1]. Visualizing inflammation with an M1 macrophage selective probe via GLUT1 as the gating target. Nat Commun. 2022 Oct 10;13(1):5974.

C39H52IN3O6

785.75

2991899-29-5

Typically exists as solids at room temperature

O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1NC(CCCC[N+]2=C(/C=C/C=C/C=C3N(CCC)C(C=CC=C4)=C4C/3(C)C)C(C)(C)C5=C2C=CC=C5)=O.[I-]

2934.99.9001

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)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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