Cargando…

Optimization and Validation of FePro Cell Labeling Method

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) inc...

Descripción completa

Detalles Bibliográficos
Autores principales: Janic, Branislava, Rad, Ali M., Jordan, Elaine K., Iskander, A. S. M., Ali, Md M., Varma, N. Ravi S., Frank, Joseph A., Arbab, Ali S.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2690694/
https://www.ncbi.nlm.nih.gov/pubmed/19517015
http://dx.doi.org/10.1371/journal.pone.0005873
_version_ 1782167846838599680
author Janic, Branislava
Rad, Ali M.
Jordan, Elaine K.
Iskander, A. S. M.
Ali, Md M.
Varma, N. Ravi S.
Frank, Joseph A.
Arbab, Ali S.
author_facet Janic, Branislava
Rad, Ali M.
Jordan, Elaine K.
Iskander, A. S. M.
Ali, Md M.
Varma, N. Ravi S.
Frank, Joseph A.
Arbab, Ali S.
author_sort Janic, Branislava
collection PubMed
description 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 µg/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 µg/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 µg/ml of Fe and 3 µg/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.
format Text
id pubmed-2690694
institution National Center for Biotechnology Information
language English
publishDate 2009
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-26906942009-06-11 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 S. PLoS One Research Article 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 µg/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 µg/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 µg/ml of Fe and 3 µg/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. Public Library of Science 2009-06-11 /pmc/articles/PMC2690694/ /pubmed/19517015 http://dx.doi.org/10.1371/journal.pone.0005873 Text en Janic et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article 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 properly credited.
spellingShingle Research Article
Janic, Branislava
Rad, Ali M.
Jordan, Elaine K.
Iskander, A. S. M.
Ali, Md M.
Varma, N. Ravi S.
Frank, Joseph A.
Arbab, Ali S.
Optimization and Validation of FePro Cell Labeling Method
title Optimization and Validation of FePro Cell Labeling Method
title_full Optimization and Validation of FePro Cell Labeling Method
title_fullStr Optimization and Validation of FePro Cell Labeling Method
title_full_unstemmed Optimization and Validation of FePro Cell Labeling Method
title_short Optimization and Validation of FePro Cell Labeling Method
title_sort optimization and validation of fepro cell labeling method
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2690694/
https://www.ncbi.nlm.nih.gov/pubmed/19517015
http://dx.doi.org/10.1371/journal.pone.0005873
work_keys_str_mv AT janicbranislava optimizationandvalidationoffeprocelllabelingmethod
AT radalim optimizationandvalidationoffeprocelllabelingmethod
AT jordanelainek optimizationandvalidationoffeprocelllabelingmethod
AT iskanderasm optimizationandvalidationoffeprocelllabelingmethod
AT alimdm optimizationandvalidationoffeprocelllabelingmethod
AT varmanravis optimizationandvalidationoffeprocelllabelingmethod
AT frankjosepha optimizationandvalidationoffeprocelllabelingmethod
AT arbabalis optimizationandvalidationoffeprocelllabelingmethod