PKH 26 is a red fluorescent dye. PKH 26 can stably bind to the lipid region of the cell membrane and emit red fluorescence (Ex/Em=551/567 nm). It is mainly used for in vitro cell labeling, in vitro cell proliferation/growth research, and in vivo and in vitro studies. Cell tracing studies.

Physicochemical Properties

Molecular Formula	C59H97IN2
Molecular Weight	961.319370031357
Exact Mass	960.669
CAS #	154214-55-8
PubChem CID	162642097
Appearance	Brown to reddish brown solid powder
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	36
Heavy Atom Count	62
Complexity	1200
Defined Atom Stereocenter Count	0
SMILES	C([N+]1=C(C(C)(C)C2C=CC=CC1=2)C=CC=C1C(C2C=CC=CC=2N1CCCCCCCCCCCCCC)(C)C)CCCCCCCCCCCCCCCCCCCCC.[I-]
InChi Key	XOOOYQALQHJRCY-UHFFFAOYSA-M
InChi Code	InChI=1S/C59H97N2.HI/c1-7-9-11-13-15-17-19-21-22-23-24-25-26-27-28-30-32-34-36-42-51-61-55-47-40-38-45-53(55)59(5,6)57(61)49-43-48-56-58(3,4)52-44-37-39-46-54(52)60(56)50-41-35-33-31-29-20-18-16-14-12-10-8-2;/h37-40,43-49H,7-36,41-42,50-51H2,1-6H3;1H/q+1;/p-1
Chemical Name	2-[(E,3E)-3-(3,3-dimethyl-1-tetradecylindol-2-ylidene)prop-1-enyl]-1-docosyl-3,3-dimethylindol-1-ium;iodide
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

First general protocol. The first step in creating a PKH 26 working solution is to dissolve 1 mg of PKH 26 in 1 mL of DMSO. This will create a 1 mM stock solution. Keep in mind that the stock solution should be stored between -20°C and -80°C in the dark, and that it should not be repeatedly frozen and thawed. 1.2 Making the PKH 26 working solution: To make a 5–10 μM working solution, dilute the stock solution 1:50 or 1:100 using serum-free cell culture medium or PBS. Note: Please change the PKH 26 working solution's concentration as necessary. 2. Suspension cells (6-well plate) 2.1 Cell staining a. Remove the supernatant after centrifuging at 1000g for three to five minutes at 4°C. Use PBS to wash twice, for five minutes each time. The density of cells is 1×106/mL. b. Incubate for 10 to 45 minutes at room temperature after adding 1 milliliter of working solution. C. Discard the supernatant after centrifuging at 400 g for three to four minutes at 4°C. d. Use PBS to wash twice, for five minutes each time. e. Re-suspend cells in PBS or serum-free culture media. observation using flow cytometry or fluorescence microscopy. 2.2 Cells that adhere a. On sterile coverslips, cultivate adherent cells. b. Aspirate extra culture medium after removing the coverslip from the medium. C. To fully submerge the cells, add 100 μL of working solution, give them a little shake, and let them sit at room temperature for five to thirty minutes. d. Wash twice, giving each wash five minutes on medium. observation using flow cytometry or fluorescence microscopy. For a year, store at -80°C with protection from light. Safety Measures 1. Kindly modify the PKH 26 working fluid concentration based on the current circumstances. 2. This product is not to be used for domestic, medical, or any other use; it is only to be used for research and development. 3. Please wear a white coat and disposable gloves for your health and safety.

References

[1]. The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity. Methods. 2003;31(2):135-142.

Solubility Data

Solubility (In Vitro)

DMSO: 1.67 mg/mL (1.74 mM)

Solubility (In Vivo)

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 Formulations (e.g. IP/IV/IM/SC) 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). 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 Formulations 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). 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.0402 mL	5.2012 mL	10.4024 mL
	5 mM	0.2080 mL	1.0402 mL	2.0805 mL
	10 mM	0.1040 mL	0.5201 mL	1.0402 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.