Dec 07, 2024

Public workspaceThymidine analog labeling of proliferating cells

DOI
https://dx.doi.org/10.17504/protocols.io.261gerdmol47/v1
  • Ashley Seifert1
  • 1University of Kentucky
Ashley Seifert
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DOI: https://dx.doi.org/10.17504/protocols.io.261gerdmol47/v1
Protocol Citation: Ashley Seifert 2024. Thymidine analog labeling of proliferating cells. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerdmol47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 06, 2024
Last Modified: December 07, 2024
Protocol Integer ID: 114530
Keywords: ASAPCRN, cycling cells with thymidine analog, thymidine analog labeling, thymidine analog, assessing cell proliferation, cycling cells before the injury, proliferating cell, cell proliferation, cells this protocol, cell proliferation in response, cycling cell, seifert lab, label, injured tissue, cell, labeling, lab, institutional animal care, proliferation
Abstract
This protocol describes the procedure used by the Seifert Lab to label cycling cells with thymidine analogs to track cell proliferation in injured tissue. EdU is used to label cycling cells before the injury, and BrdU after, thereby assessing cell proliferation in response to injury. It has been successfully used in rodents (Mus musculus and Acomys dimidiatus - spiny mouse), zebrafish (Danio rerio) and axlotl (Ambystoma mexicanum).

Note that any protocol involving animals should be reviewed and approved by your Institutional Animal Care and Use Committee (IACUC) before use.
Materials
  1. Thymidine analogs diluted in DMSO (EdU and BrdU)
  2. Sterile saline solution (0.9% NaCl)
  3. 1.5 mL Eppendorf tubes
  4. Syringe (2 mL capacity, suitable gauge for injection)
  5. Appropriate injection needles (e.g., 25-30 gauge for intraperitoneal injection)
  6. Animals, e.g. Acomys dimidiatus (spiny mice), Mus musculus, Ambystoma mexicanum (axolotls), Danio rerio (zebrafish)
Protocol materials
ReagentBrdU (5-Bromo-2´-Deoxyuridine)Thermo FisherCatalog #B23151
ReagentEdU (5-ethynyl-2’-deoxyuridine)Thermo FisherCatalog #A10044
Troubleshooting
For Acomys, Mus, or Ambystoma:
Dilute ReagentEdU (5-ethynyl-2’-deoxyuridine)Thermo FisherCatalog #A10044 from stock in sterile saline to a final concentration of Concentration10 μg/g body weight

For Acomys, Mus, or Ambystoma:
Dilute ReagentBrdU (5-Bromo-2´-Deoxyuridine)Thermo FisherCatalog #B23151 from stock in sterile saline to a final concentration of Concentration1 μg/g body weight

For Acomys, Mus, or Ambystoma:
Transfer the thymidine analog solution to a 2 mL syringe appropriate for intraperitoneal injection (25-30 gauge)
For Acomys, Mus, or Ambystoma:
Inject EdU intraperitoneally three times at 3-hour intervals (i.e., 0h, 3h, and 6h)
For Acomys, Mus, or Ambystoma:
Collect tissue at 9h (for assessment of baseline cell proliferation)
For Acomys, Mus, or Ambystoma:
At desired time following injury, inject BrdU intraperitoneally three times at 3-hour intervals (i.e., 0h, 3h, and 6h)
For Acomys, Mus, or Ambystoma:
Collect tissue at 9h (for assessment of injury-induced cell proliferation)
For Danio:
Prepare a final solution of Concentration0.4 millimolar (mM) EdU or BrdU in zebrafish water

Optional
For Danio:
Place zebrafish in the prepared water for a Duration09:00:00 exposure period to allow analog uptake before collecting tissue

Optional