Log In Sign up
The largest database of trusted experimental protocols

Pkh26 labeling kit

Manufactured by Merck Group
Visit Supplier
Sourced in Macao

The PKH26 labeling kit is a fluorescent cell linker that can be used to label cells for tracking and in vitro cell-based assays. The kit provides the necessary reagents to effectively label cells with the PKH26 red fluorescent dye.

Automatically generated - may contain errors

Lab products found in correlation

Robosep buffer, by STEMCELL (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Zetaview pmx 120, by Particle Metrix (1 mentions) Jem 1010, by JEOL (1 mentions) Stellaris sted, by Leica (1 mentions) Type 90 ti, by Beckman Coulter (1 mentions) Transmission electron microscope, by Hitachi (1 mentions) Exoquick tctm exosome precipitation solution, by System Biosciences (1 mentions) Vivacon 500, by Sartorius (1 mentions)

Spelling variants of this product

PKH26 (838 citations) PKH26 kit (31 citations) PKH26 red fluorescent labeling kit (20 citations) PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (16 citations) PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (5 citations) PKH26 fluorescent labeling kit (5 citations) PKH26 red fluorescent cell membrane labeling kit (4 citations) PKH26 Red Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (4 citations) PKH26 red fluorescence labeling kit (2 citations)

8 protocols using pkh26 labeling kit

1

Monocyte Isolation and PKH26 Labeling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Bone marrow was harvested by flushing the femurs and tibia with RoboSep Buffer (Stemcell Tech, Vancouver, BC, Canada) using a syringe with a 27-gauge needle. Cell clumps were removed by passing the cell suspension through a 70-μm mesh nylon strainer and the cells were centrifuged at 300 × g for 6 min. The pelleted cells were resuspended at 1 × 108 cells/ml, and monocytes were enriched with an EasySep Mouse Monocyte Enrichment kit according to the manufacturer’s instructions.
Isolated monocytes (5 × 105 to 1 × 106) were labeled with PKH26 using a PKH26 Labeling kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. PKH26 fluorescence (a yellow-orange fluorescent dye with long aliphatic tails) technology provides stable incorporation into lipid regions of cell membranes and has been found to be useful for in vitro and in vivo cell tracking applications in a wide variety of systems. In brief, cells were washed once in serum-free RPMI-1640 medium, resuspended in 2 mL kit diluent solution C, mixed with PKH26 at 2 × 10−3 mol/L in diluent C, and incubated for 10 min at room temperature in the dark. An equal volume of medium containing 10% FBS was added, and the cells were centrifuged, washed once, and resuspended in serum-containing medium for further analysis.
Ikeda Y., Sonoda N., Bachuluun B., Kimura S., Ogawa Y, & Inoguchi T. (2020). Aberrant activation of bone marrow Ly6C high monocytes in diabetic mice contributes to impaired glucose tolerance. PLoS ONE, 15(2), e0229401.
+ Open protocol
+ Expand
2

Internalization and Efferocytosis Assays

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Bacterial internalization assays were performed as previously described (7 (link)), using opsonized Serotype 14 S. pneumoniae (National Collection of Type Cultures 11902). For efferocytosis assays, neutrophils isolated via Percoll gradient were stained with a PKH26 labeling kit, as per manufacturer’s guidelines (Sigma-Aldrich), and cultured for 20 hours in RPMI 1640. Apoptotic neutrophils (70–80% apoptotic verified by Annexin-V-Topro3 staining) were added to macrophages at MOI 5:1, with ice control, for 90 minutes before vigorous washing and removal for analysis on a Becton Dickinson FACS Calibur flow cytometer (20 (link)).
Ryan E.M., Sadiku P., Coelho P., Watts E.R., Zhang A., Howden A.J., Sanchez-Garcia M.A., Bewley M., Cole J., McHugh B.J., Vermaelen W., Ghesquiere B., Carmeliet P., Rodriguez Blanco G., Von Kriegsheim A., Sanchez Y., Rumsey W., Callahan J.F., Cooper G., Parkinson N., Baillie K., Cantrell D.A., McCafferty J., Choudhury G., Singh D., Dockrell D.H., Whyte M.K, & Walmsley S.R. (2023). NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine, 207(8), 998-1011.
+ Open protocol
+ Expand
3

Fluorescent Labeling of Extracellular Vesicles

Check if the same lab product or an alternative is used in the 5 most similar protocols
For immunofluorescent studies, sEVs were labeled with general lipophilic fluorescent membrane dye, PKH26, using the PKH26 labeling kit according to manufactures instructions (Sigma-Aldrich). Briefly, purified aliquots of 20 μg sEVs were resuspended in 180 μl diluent C and mixed with 20 μl PKH26 dye diluted in diluent C for 5 min. Staining was stopped by washing labeled exosomes with 0.1% BSA in nuclease free water and pelleted by centrifugation at 110,000 x g for 70 min at 4°C. Labeled sEVs pellet was resuspended in 50 μl PBS and stored in −80°C until use.
Jean-Toussaint R., Lin Z., Tian Y., Gupta R., Pande R., Luo X., Hu H., Sacan A, & Ajit S.K. (2021). Therapeutic and prophylactic effects of macrophage-derived small extracellular vesicles in the attenuation of inflammatory pain. Brain, behavior, and immunity, 94, 210-224.
+ Open protocol
+ Expand
4

In vivo Monocyte Tracking in Mice

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
In vivo monocyte tracking was performed as previously described42 (link). Briefly, leukocytes pooled from 10-wk-old male C57BL/6 mice bled from the retro-orbital sinus were subjected to red blood cell lysis. Isolated monocytes were washed in RPMI-1640 medium, counted, and suspended in 2 ml diluent solution C included in the PKH26 labeling kit (Sigma, St. Louis, MO) per 5 × 106 to 10 × 106 cells. 2 ml PKH26 diluted to 2 × 10−3 mol/L in diluent solution C was added to the monocytes and mixed. Monocytes were incubated in the PKH26-containing solution for 10 min in the dark at room temperature. Staining was halted by the addition of medium containing 10% FBS and monocytes washed in PBS and resuspended in serum containing medium. PKH26-labeled monocytes were counted and 1 × 106 cells were suspended in 0.2 ml PBS and injected retro-orbitally into each recipient mouse (HFD-fed fl/fl or PU.1 AKO mice). SVCs were isolated from recipient mice 5 days post injection, eWAT, stained, and analyzed by FACS.
Lackey D.E., Reis F.C., Isaac R., Zapata R.C., El Ouarrat D., Lee Y.S., Bandyopadhyay G., Ofrecio J.M., Oh D.Y, & Osborn O. (2019). Adipocyte PU.1 knockout promotes insulin sensitivity in HFD-fed obese mice. Scientific Reports, 9, 14779.
+ Open protocol
+ Expand
5

Isolation and Characterization of Small Extracellular Vesicles

Check if the same lab product or an alternative is used in the 5 most similar protocols
sEVs were extracted by sequential ultracentrifugation: briefly, cells culture was replaced by medium containing 10% of EV‑depleted FBS. Then, cell culture medium was collected and centrifuged at 300 × g for 10 min, 2000 × g for 15 min, and finally at 12,000 × g for 30 min to remove cell debris. After filtration through 0.22-µm filters, the supernatant was ultracentrifuged at 100,000 × g (Beckman Type 90 Ti) for 2 h. The sEVs pellets were washed in PBS and ultracentrifuged at 100,000 × g for 2 h again. All samples were re-suspended in PBS for further analyses.
The isolated sEVs were absorbed by carbon coated copper networks for 2 min, washed in ddH2O, and finally negative staining with uranium acetate for 2 min. All samples were observed by Transmission electron microscopy (TEM) (JEM-1010, JEOL, Japan). The size distributions and concentrations of diluted sEVs were analyzed by Nanoparticle Tracking Analyzer (ZetaView®PMX120, Particle Metrix, German).
sEVs were labeled with a PKH26 Labeling Kit (Sigma-Aldrich, Saint Louis, USA) following the manufacturer’s instructions. The uptake of PKH26-labeled sEVs by macrophages was examined using confocal microscopy (Stellaris STED, LEICA, Germany).
Xu D., Chen W.Q., Liang M.X., Chen X., Liu Z., Fei Y.J., Shao X.Y., Wu Y., Zhang W, & Tang J.H. (2023). Tumor-derived small extracellular vesicles promote breast cancer progression by upregulating PD-L1 expression in macrophages. Cancer Cell International, 23, 137.
+ Open protocol
+ Expand
6

Isolation and Characterization of ADSC-Derived Exosomes

Check if the same lab product or an alternative is used in the 5 most similar protocols
ADSCs were trypsinized and seeded at 5 × 105 cells in a 10-cm dish. After 24 h, the culture medium was collected and centrifuged at 3,000 g for 15 min to remove cells and cell debris. ADSC-Exo were isolated from 10 mL culture media using 2 mL ExoQuick-TCTM Exosome Precipitation Solution per the manufacturer’s instructions (System Biosciences, CA, United States). After overnight incubation at 4°C, the mixture was centrifuged at 10,000 g for 30 min at 4°C. The pellet was washed with 1 mL PBS and centrifuged at 4°C for 15 min at 10,000 g. Then, the purified ADSC-Exos were completely suspended in 50 μL PBS and stored at −80°C for further study. The protein content of the exosomes was quantified using the Bradford method. The purified ADSC-Exo was characterized using the biomarker CD63 and CD9 by Western blot. The morphology and size of ADSC-Exo were observed under a transmission electron microscope (Hitachi, Tokyo, Japan). Nanoparticle tracking analysis (NTA) was used to evaluate the concentration and size distribution of ADSC-Exo. The detailed methods and results for characterization of ADSC-Exo were described in the Supplementary Data. Cardiac and cellular uptake of ADSC-Exo were examined using the PKH-26 labeling kit (Sigma, MO, United States) and observed under a confocal microscope.
Lai T.C., Lee T.L., Chang Y.C., Chen Y.C., Lin S.R., Lin S.W., Pu C.M., Tsai J.S, & Chen Y.L. (2020). MicroRNA-221/222 Mediates ADSC-Exosome-Induced Cardioprotection Against Ischemia/Reperfusion by Targeting PUMA and ETS-1. Frontiers in Cell and Developmental Biology, 8, 569150.
+ Open protocol
+ Expand
7

Exosome Labeling with PKH26 Dye

Check if the same lab product or an alternative is used in the 5 most similar protocols
For labeling experiments, exosome pellets were resuspended directly in diluent C from the lipophilic PKH‐26 labeling kit (Sigma). Labelling was performed following the manufacturer's instructions, with the following modifications for exosome labelling. To stop the reaction, 2% BSA was used, as FBS contains endogenous exosomes. To remove unbound dye, preparations were loaded on to a 7 kDa Zeba spin column (Pierce). A PKH labelling control was obtained by putting a tube with diluent alone through the PKH staining procedure. This was used in subsequent experiments by adding the same volume as required for the concentration of the corresponding exosome sample (designated PKH control).
Holder B., Jones T., Sancho Shimizu V., Rice T.F., Donaldson B., Bouqueau M., Forbes K, & Kampmann B. (2016). Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal–Placental Messaging. Traffic (Copenhagen, Denmark), 17(2), 168-178.
+ Open protocol
+ Expand
8

Exosome Labeling and Cell Uptake

Check if the same lab product or an alternative is used in the 5 most similar protocols
Exosomes were labeled with the fluorescent dye PKH-26 using the PKH-26 labeling kit (Sigma-Aldrich Co.) [22] . Briefly, 0 Gy-Exo and 5 Gy-Exo were labeled with 2 μM PKH-26 for 15 min at room temperature. Thereafter, free PKH-26 was removed by ultrafiltration using the VIVACON 500 ultracentrifugation device (100,000 MWCO; Sartorius Stedim Biotech GmbH, Goettingen, Germany). The labeled exosomes were added to MIAPaCa-2 cell samples and the nuclei of cells were stained with Hoechst 33342 for 5 min.
Nakaoka A., Nakahana M., Inubushi S., Akasaka H., Salah M., Kubota H., Fujita Y., Hassan M., Mukumoto N., Nishikawa R., Ishihara T., Miyawaki D., Yoshida K., Sasayama T., & Sasaki R. (2020). Exosome-mediated radiosensitizing effect on neighboring cancer cells via reactive oxygen species production.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!