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Kim-1 targeting ability

XL Xuefang Lou
SJ Saiping Jiang
YD Yongzhong Du

Last updated date: Feb 26, 2025 Views: 344 Forks: 0

original An abbreviated version of this protocol was published in Science Advances in Oct, 2020
ROS-responsive chitosan-SS31 prodrug for AKI therapy via rapid distribution in the kidney and long-term retention in the renal tubule
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  1. HK-2 cells were seeded at a density of 1×10⁵ cells/well in 12-well plates containing glass coverslips and cultured overnight to facilitate cell adhesion.
  2. For oxidative stress induction, H2O2 was introduced into the culture medium at a final concentration of 500 μM, followed by 4-hour incubation. H2O2  was allowed to pre-mixed with fresh culture medium to achieve the desired concentration (500 μM) before complete medium replacement.
  3. KIM-1 receptor blockade was achieved by incubating HK-2 cells with KIM-1 antibody (Novus Biologicals, USA) at a final concentration of 30 μg/mL for 30 minutes. As a control, parallel cultures of H2O2-stimulated HK-2 cells were maintained without KIM-1 antibody treatment.
  4. Following receptor blockade, cells were incubated with either Cy5-labeled Ser-CS or Cy5-labeled CS at a final concentration of 500 μg/mL for an additional 4-hour period.
  5. Post-incubation, the culture medium was aspirated, and cells were subjected to two washes with 1×PBS, fixed with 4% formaldehyde for 10 minutes at room temperature, and subsequently washed three times with 1×PBS.
  6. For immunostaining, cells were incubated with KIM-1 antibody (1:200 dilution) at 4°C overnight, followed by three PBS washes to remove unbound antibody.
  7. Detection was performed using FITC-conjugated goat anti-rabbit IgG secondary antibody with a 2-hour incubation at room temperature, followed by three PBS washes. Nuclear staining was accomplished through 20-minute incubation with DAPI at room temperature, with subsequent PBS washes.
  8. Finally, the glass coverslips were mounted using glycerol and subjected to confocal laser scanning microscopy (CLSM) for image acquisition.
Copyright: © 2025 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Lou, X, Jiang, S and Du, Y(2025). Kim-1 targeting ability. Bio-protocol Preprint. bio-protocol.org/prep2793.
  2. Liu, D., Shu, G., Jin, F., Qi, J., Xu, X., Du, Y., Yu, H., Wang, J., Sun, M., You, Y., Zhu, M., Chen, M., Zhu, L., Shen, Q., Ying, X., Lou, X., Jiang, S. and Du, Y.(2020). ROS-responsive chitosan-SS31 prodrug for AKI therapy via rapid distribution in the kidney and long-term retention in the renal tubule . Science Advances 6(41). DOI: 10.1126/sciadv.abb7422

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