Optimization and validation of FePro cell labeling method

Branislava Janic, Ali M. Rad, Elaine K. Jordan, A. S.M. Iskander, Md M. Ali, N. Ravi S. Varma, Joseph A. Frank, Ali Syed Arbab

Research output: Contribution to journalArticle

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Abstract

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 mg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 mg/ml and Pro 0.75 to 3 μg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30-35 pg-iron/cell at 24 h when labeled with 100 μg/ml of Fe and 3 μg/ ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 μg/ml of Fe and 3 μg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 mg/ml of Fe and 3 mg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 μg/ml of Fe and 3 μg/ml of Pro is effective in labeling cells for cellular MRI.

Original languageEnglish (US)
Article numbere5873
JournalPloS one
Volume4
Issue number6
DOIs
StatePublished - Jun 11 2009
Externally publishedYes

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Protamines
Labeling
protamines
cells
methodology
Iron
Stem cells
Magnetic resonance imaging
iron
Cells
cell viability
Cell Survival
dosage
T-cells
Serum-Free Culture Media
ferumoxides
labeling techniques
stromal cells
bone marrow cells
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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Janic, B., Rad, A. M., Jordan, E. K., Iskander, A. S. M., Ali, M. M., Varma, N. R. S., ... Arbab, A. S. (2009). Optimization and validation of FePro cell labeling method. PloS one, 4(6), [e5873]. https://doi.org/10.1371/journal.pone.0005873

Optimization and validation of FePro cell labeling method. / Janic, Branislava; Rad, Ali M.; Jordan, Elaine K.; Iskander, A. S.M.; Ali, Md M.; Varma, N. Ravi S.; Frank, Joseph A.; Arbab, Ali Syed.

In: PloS one, Vol. 4, No. 6, e5873, 11.06.2009.

Research output: Contribution to journalArticle

Janic, B, Rad, AM, Jordan, EK, Iskander, ASM, Ali, MM, Varma, NRS, Frank, JA & Arbab, AS 2009, 'Optimization and validation of FePro cell labeling method', PloS one, vol. 4, no. 6, e5873. https://doi.org/10.1371/journal.pone.0005873
Janic B, Rad AM, Jordan EK, Iskander ASM, Ali MM, Varma NRS et al. Optimization and validation of FePro cell labeling method. PloS one. 2009 Jun 11;4(6). e5873. https://doi.org/10.1371/journal.pone.0005873
Janic, Branislava ; Rad, Ali M. ; Jordan, Elaine K. ; Iskander, A. S.M. ; Ali, Md M. ; Varma, N. Ravi S. ; Frank, Joseph A. ; Arbab, Ali Syed. / Optimization and validation of FePro cell labeling method. In: PloS one. 2009 ; Vol. 4, No. 6.
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