M. Berger and Bamdad participated in research design.
Version of Record online: 28 AUG 2012
Copyright © 2012 International Society for Advancement of Cytometry
Cytometry Part A
Volume 81A, Issue 11, pages 996–1004, November 2012
How to Cite
Bourgne, C., Bamdad, M., Janel, A., Libert, F., Gagnieu, M.-C., Rapatel, C., Pigeon, P., Pereira, S., Hermet, E., Guerci, A., Pereira, B., Makhoul, P. C., Ansah, A. J., Cahn, J.-Y., Guyotat, D., Trouillier, S., Berger, J., Boiret-Dupré, N. and Berger, M. G. (2012), Measurement of imatinib uptake by flow cytometry . Cytometry, 81A: 996–1004. doi: 10.1002/cyto.a.22118
Bourgne, Pereira, Pigeon, J. Berger, Rapatel, and Gagnieu conducted experiments.
M. Berger, Bourgne, and Bamdad performed data analysis.
M. Berger, Libert, Boiret-Dupré, Janel Bourgne, and Bamdad wrote or contributed to the writing of the manuscript.
M. Berger, Hermet, Guerci, Cony-Makhoul, Johnson Ansah, Cahn, Guyotat and Trouillier provided primary cells.
- Issue online: 22 OCT 2012
- Version of Record online: 28 AUG 2012
- Manuscript Accepted: 17 JUL 2012
- Manuscript Received: 10 NOV 2011
- GECOM (Groupe d'Etude des Cellules Mésenchymateuses) Association and the Clermont-Ferrand University Hospital Center
- French National Center for Scientific Research (Centre National de la Recherche Scientifique)
Additional Supporting Information may be found in the online version of this article.
|CYTO_22118_sm_SuppTab1.tif||5288K||Table S1: UV fluorescent calibrated beads tested to standardize ICIM measurement. Several commercialized beads were tested to ensure flow cytometer stability during analysis. Only a few beads are designed to have a specific UV application, and they did not correspond to our experimental conditions. We also tested FITC fluorescent beads and chose the Flow Check Fluorosphere whose fluorescence in linear scale was homogeneous and located in the area of IM fluorescence.|
|CYTO_22118_sm_SuppFig1.tif||4563K||Figure S1: UV fluorescence of marketed calibrated beads. Different calibrated beads were analyzed through UV laser excitation. In most cases, the signal obtained was neither homogeneous nor accurate enough (large peak), or not in the space corresponding to fluorescence intensity emitted from one cell containing IM requiring projection on linear scale. Demonstrative examples in (A) ImmunobriteTM, (B) Cyto-CalTM, (C) SHEROTM Rainbow, (D) SpheroTM UV Carboxyl Particle. Only Flow Check FluorospshereTM (E, F) met our criteria to calibrate the cytometer before each experiment; their UV fluorescence intensity was uniform and stable over time and within the space of IM fluorescence. For each tested beads, the CV is indicated next to the peak.|
|CYTO_22118_sm_SuppFig2.tif||4881K||Figure S2: UV spectrum of IM (A) and demonstrative example of sample chromatogram obtained with a solution used in the set of standards and containing 0.150omg/L of imatinib and its metabolite N-desmethyl imatinib at a wavelength of 264onm (B).|
|CYTO_22118_sm_SuppFig3.tif||4917K||Figure S3: The ICIM level in cell lines is not related to membrane pump expression. We evaluated, in parallel, membrane pump expression and the ability of K562 and KCL22 cells to store IM. Total RNA was isolated from cells using the Nucleospin RNA II kit (Macherey Nagel); cDNA was synthetized using the cDNA High Capacity Reverse Transcription kit, according to the manufacturer's instructions. A TaqMan quantitative real time PCR was then performed on a RotorGene 6000 real time thermal cycler (Corbett Biosciences) using TaqMan Gene Expression Assays (Hs00427552_m1 for OCT-1, Hs01053790_m1 for ABCG2, Hs01067802_m1 for ABCB1, Hs00219905_m1 for ABCC1, Hs00984230_m1 for β2-microglobuline; Applied Biosystems). The amount of target transcript was analyzed using the 2-ΔΔ Ct method and was normalized to the endogenous reference gene (β2-microglobuline) and K562 cells as the calibrator (n=3) The relative expression of the membrane pump in KCL22 cells are 0.32±0.1 for OCT-1, 0.31±0.12 for ABCG2, 1.36±0.4 for ABCC1 and 0.05±0.006 for ABCB1, according to the expression in K562 cells used as calibrator (arbitrary value: 1) (A); hOCT-1 expression was also analyzed at the protein level using western blot. Briefly, K562 and KCL22 cells were lyzed in an extraction protein buffer supplemented with protease inhibitors (Roche Diagnostics), and 30µg of protein were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were incubated with diluted antibody (anti-OCT1, Aviva Systems Biology, 2.5µg/ml, and anti-tubulin, Ozyme) overnight at 4oC with gentle shaking. After incubation with appropriate secondary antibody for 1 hr at room temperature, immunoblots were visualized with an ECL system (ECL prime Western Blot Detection Reagent Kit, VWR). (B): ICIM level in K562 and KCL22 cell lines after 1 hr of incubation with 1 µM IM (n=5) (C).|
|CYTO_22118_sm_SuppFig4.tif||4888K||Figure S4: Demonstrative example of flow cytometric analysis of apoptotic K562 cells. K562 control cells (A) and K562 treated by 25µM of IM for 24 hrs (B) were stained with PI and Annexin-V. Viable cells are visualized in B3 and A3 quadrant (PI-/AnnexinV-).|
|CYTO_22118_sm_SuppFig5.tif||4767K||Figure S5: The active metabolite N-Desmethyl IM remains detectable by flow cytometry. N-Desmethyl IM, the main active metabolite of IM, could be detected by flow cytometry using UV-laser equipped-cytometer. K562 and KCL22 cell lines were incubated for 1 hr with 0.5/1/5/10/25 or 50 µM. We then applied the protocol used to detect IM to follow N-Desmethyl IM uptake in cells. We observed a similar dose-response curve as with IM; at lower extra-cellular concentrations (0.5 and 1 µM) of N-Desmethyl IM, the uptake was significantly higher in KCL 22 cells (**p<0.01). Results are expressed as mean ± SEM, n=5.|
|MIFlowCyt-Item-Location.doc||46K||Supporting Information: MIFlowCyt|
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