For immunofluorescence Staining, the mainly procedure was described in previously report (Zhang et al., 2022 (link)). Briefly, the treated Hela/DDP and Caski/DDP cells were washed by PBS and fixed by ice chilled methanol. The exosome was labeled by PKH26 (Puzar Dominkus et al., 2018 (link)) using PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich). DAPI (Sigma-Aldrich) stained cellular nucleus with following the instruction, slides were imaged by confocal laser scanning microscopy (Olympus).
Pkh26 red fluorescent cell linker kits for general cell membrane labeling
Manufactured by Merck Group
Sourced in United States
PKH26 Red Fluorescent Cell Linker Kits are a set of reagents used for general cell membrane labeling. The kits provide a red fluorescent dye that binds to the cell membrane, allowing for the visualization and tracking of cells.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using pkh26 red fluorescent cell linker kits for general cell membrane labeling
1
Immunofluorescent Exosome Tracking in Cells
2
Labeling of Nf-EVs for Uptake Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The isolated Nf-EVs were stained with red fluorescent dye PKH26 (Sigma-Aldrich, St. Louis, MO, USA) using the PKH26 red fluorescent cell linker kits for general cell membrane labeling (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, with modification. Initially, 6 µL of PKH26 dye was diluted in 1.5 mL of diluent C. Then 30 µL of Nf-MP and Nf-Exo were diluted in 500 µL of diluent C and gently mixed with dye solution (1:1 v/v) for 5 min. The labeling reaction was stopped by adding an equal volume of FBS for 1 min. Next, PKH26 labeled EVs were diluted with 10 mL of complete medium and centrifuged at 21,000× g for 70 min at 4 °C to pellet the PKH26-labeled MP (MP-PKH26) and centrifuged at. 110,000× g for 90 min at 4 °C to pellet the PKH26-labeled exosomes (Exo-PKH26). The PKH26 labeled EVs were further washed with complete medium following the same procedure to remove any free dye, and finally, the MP-PKH26 and Exo-PKH26 were obtained and resuspended in 600 µL of complete medium and used for EVs uptake studies. For the control, labeling was performed as described but without EVs.
3
Morphological Characterization of A-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monolayer and 3D spheroid A-MSCs were subjected to scanning electron microscopy by WeiYa Bio-Technology Co., Ltd. (Jinan, Shandong, China). Samples were fixed using 2.5% glutaraldehyde solution for 3 h at 4°C. Microstructural characterization was performed using a high-resolution scanning electron microscope (Sigma300; Zeiss, Germany).
To observe the overall distribution of cells, digested A-MSCs were first stained with PKH26 using PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich, St Louis, MO, USA). Prior to staining, PKH26 in diluent C was incubated in a water bath at 37 °C for 15 min. A-MSCs stained with a cell membrane were cultured by the hanging drop method. After 24 h incubation in a 37°C incubator, the original position of the suspension was maintained for 4′,6-diamidino-2-phenylindole (DAPI) staining. Next, 3D spheroid A-MSCs were subjected to hematoxylin and eosin staining for morphological evaluation. The spheroids were observed under a microscope (BX53; Olympus, Tokyo, Japan) and photographed (CellSens, Ver. 1.18; Tokyo, Japan).
To observe the overall distribution of cells, digested A-MSCs were first stained with PKH26 using PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich, St Louis, MO, USA). Prior to staining, PKH26 in diluent C was incubated in a water bath at 37 °C for 15 min. A-MSCs stained with a cell membrane were cultured by the hanging drop method. After 24 h incubation in a 37°C incubator, the original position of the suspension was maintained for 4′,6-diamidino-2-phenylindole (DAPI) staining. Next, 3D spheroid A-MSCs were subjected to hematoxylin and eosin staining for morphological evaluation. The spheroids were observed under a microscope (BX53; Olympus, Tokyo, Japan) and photographed (CellSens, Ver. 1.18; Tokyo, Japan).
4
Quantifying NDMV Uptake by FLS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To aid the quantification of the number of NDMVs taken up by FLS, the phospholipid bilayer membrane of the NDMVs was stained by PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich, St. Louis, MO, USA). NDMV pellets obtained after ultra-centrifugation were first resuspended gently in 100 μL Diluent C. Equivalent 2×Dye Solution (PKH26 solution/Diluent C ratio of 4 μL:1000 μL) was added to the NDMV mixture and incubated for 5 min at room temperature (RT). An amount of 200 μL EV-free serum was used to stop staining for 1 min at RT. An additional 5 mL RPMI 1640/10% EV-free serum was applied to wash the PKH26-stained NDMVs and dilute residual PKH26. Ultra-centrifugation was used to pellet PKH26-stained NDMVs (Figure S4B ) at 100,000× g for 30 min at 4 °C which were suspended in 100 μL EV-free RPMI 1640. After the size distribution and concentration of PKH26-labeled NDMVs were verified, FLS were treated with NDMVs with an appropriate concentration to measure internalization using flow cytometry and confocal microscopy.
5
Tracking Tumor Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions of 2 × 106 cells were prepared and labeled with PKH26 dye in diluent C (1:500 dilution, yielding a final concentration of 2 μmol/L) as per instructions for PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma‐Aldrich). The cells were washed twice to remove any unbound dye and then suspended in MEBM with supplements at the density of 6 × 104 cells/well in a 6‐well low‐adherent plate. Tumorspheres were collected at different time (day) intervals, centrifuged, and dissociated into a single cell suspension with Accumax/D‐PBS (1:1) prior to analysis with a Gallios flow cytometer (Beckman Coulter, Inc. CA, USA). Proliferation index (PI) and various other cell tracking parameters were studied and quantified using Modfit LT V4.1.7 software with the cell tracking wizard module.
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
Revolutionizing how scientists
search and build protocols!