Figure 4.
CSF does not influence methotrexate uptake and retention by leukemia cells. (A-C) Trimetrexate dose-response curves for NALM-6 (A), REH (B), and Jurkat (C) leukemia cells treated in either regular media or CSF. Leukemia cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. (D-E) Leukemia cells were treated with talotrexin 1 μm (D) or piritrexim 1 μM (E) in either regular tissue culture media or CSF. Leukemia cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001 by t test. (F-H) Fluorescent methotrexate retention in leukemia cells. NALM-6 (F), REH (G), and Jurkat (H) cells loaded with fluorescein-conjugated methotrexate were treated with unlabeled methotrexate 500 nM or DMSO in regular media or CSF. Leukemia cell fluorescence was then measured by flow cytometry at 0, 30, 45, 60, 90, and 120 minutes. Error bars represent the mean ± SD of 3 technical replicates. (I) Methotrexate polyglutamate species 2-4 were measured using LC-MS/MS in Jurkat leukemia cells after treatment with methotrexate 215 nM for 24 hours in either regular media or CSF. ns, not significant by Student t test.

CSF does not influence methotrexate uptake and retention by leukemia cells. (A-C) Trimetrexate dose-response curves for NALM-6 (A), REH (B), and Jurkat (C) leukemia cells treated in either regular media or CSF. Leukemia cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. (D-E) Leukemia cells were treated with talotrexin 1 μm (D) or piritrexim 1 μM (E) in either regular tissue culture media or CSF. Leukemia cell viability was assessed after 48 hours of drug treatment using the CellTiter-Glo luminescent cell viability assay. Error bars represent the mean ± SD of 3 technical replicates. ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001 by t test. (F-H) Fluorescent methotrexate retention in leukemia cells. NALM-6 (F), REH (G), and Jurkat (H) cells loaded with fluorescein-conjugated methotrexate were treated with unlabeled methotrexate 500 nM or DMSO in regular media or CSF. Leukemia cell fluorescence was then measured by flow cytometry at 0, 30, 45, 60, 90, and 120 minutes. Error bars represent the mean ± SD of 3 technical replicates. (I) Methotrexate polyglutamate species 2-4 were measured using LC-MS/MS in Jurkat leukemia cells after treatment with methotrexate 215 nM for 24 hours in either regular media or CSF. ns, not significant by Student t test.

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