Name: Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS (Video) | JoVE Journal
Uploaded: 2026-01-23T13:30:00.000+00:00
Duration: 8 min 24 s
Description: Learn LC-MS/MS quantification of vincristine in pediatric ALL cells. This validated method enables precise intracellular drug measurement to optimize therapeutic monitoring and improve treatment outcomes.

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We thank the Centro Infantil Boldrini and all the patients who donated their samples for the study. Adrielli C. Soares received a fellowship from the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES) and from the S&#227;o Paulo Research Foundation (FAPESP, 2023/18389-3). Jos&#233; A. Yunes received a productivity fellowship from the National Counsel of Technological and Scientific Development (CNPq, 308399/2021-8). This work was supported by research funding from the Brazilian Ministry of Health by the PRONON program (Programa Nacional de Apoio &#224; Aten&#231;&#227;o Oncol&#243;gica, NUP 25000.211174/2019-45).

The leukemia samples used in this study were obtained from bio-banked vials of patients treated at Centro Infantil Boldrini, who allowed the use of their cells through an informed consent form. The projet was approved by the Institutional Ethics Committee (CAAE 34601120.7.0000.5376). The protocols for the generation of PDX in animals were carried out in accordance with the regulations and ethical guidelines of the Animal Ethics Committee (Comiss&#227;o de &#201;tica no Uso de Animais) of Centro Infantil Boldrini (CEUA/Boldrini 0011-2020).
1. Sample preparation and drug incubation (Day 1)
<ol>
	<li>Preparation of stock and working solutions
	<ol>
		<li>Weigh 2.5 mg of Vincristine sulfate (molecular weight: 923 g/mol) and dissolve it in 2.0 mL of dimethyl sulfoxide (DMSO) in a 2.0 mL microtube to prepare a 1.354 mM stock solution. Vortex until fully dissolved. 
		&#8203;CAUTION: DMSO is an irritant; wear gloves and handle it in a chemical fume hood.</li>
		<li>Prepare the 100 &#181;M working solution by mixing 147.71 &#181;L of the VCR stock solution with 1,852.28 &#181;L of DMSO in a 2.0 mL microtube. Vortex thoroughly.</li>
		<li>Distribute 500 &#181;L of the working solution into four 0.6 mL microtubes and store at -20 &#176;C for up to several months. Avoid thawing the aliquots more than three times.</li>
		<li>Prepare a 100 &#181;M vinblastine (VINB) working solution by mixing 181.81 &#181;L of a 1.1 mM stock solution (Vinblastine sulfate 1 mg/mL, molecular weight: 909.07 g/mol) with 1,818.19 &#181;L of sterile ultrapure water in a 2.0 mL microtube. Vortex until homogeneous.</li>
		<li>Aliquot the VINB working solution the same way as done for VCR.</li>
		<li>Prepare methanol + 0.1 &#181;M VINB solution by mixing 49.95 mL of methanol with 50 &#181;L of the VINB working solution in a 50 mL tube. Vortex until homogeneous. Store it at -20 &#176;C up to 3 months. 
		CAUTION: Methanol is toxic and flammable; handle with appropriate PPE in a fume hood.</li>
		<li>Prepare a water + 0.1% formic acid solution by mixing 49.95 mL of ultrapure water with 50 &#181;L of formic acid in a 50 mL tube. Vortex well. Store at room temperature (RT). 
		CAUTION: Formic acid is corrosive; handle with gloves and eye protection.</li>
	</ol>
	</li>
	<li>Thawing and washing primary cells
	<ol>
		<li>Thaw cryopreserved cell samples by gently swirling the vial in a beaker containing 15 mL of 37 &#176;C distilled water until fully thawed. 
		&#8203;NOTE: Information regarding the cell collection and freezing protocols can be found in previous publications9,10.</li>
		<li>Transfer the contents of the vial to a 50 mL conical tube containing 10-15 mL of sterile 1x PBS with a sterile disposable Pasteur pipette. Centrifuge at 300 &#215; g for 5 min at RT.</li>
		<li>Discard the supernatant by inverting the tube.</li>
		<li>Resuspend the pellet in 10 mL of AIM V medium with a disposable Pasteur pipette. Count viable cells and measure average cell size using an automated counter. 
		NOTE: Using cells that exhibit at least 80% viability relative to the total number of cells collected is recommended.</li>
		<li>Calculate the volume needed to collect 2.5 &#215; 106&#160;viable cells using a simple proportion based on the measured concentration. For example, if the concentration is 5 &#215; 106&#160;cells/mL, the required volume is 0.5 mL. 
		&#8203;NOTE: Perform each treatment in triplicate for data reproducibility. Save the medium size of the cells provided in the automated cell counter; use it to calculate the volume of the cells.</li>
	</ol>
	</li>
	<li>Drug treatment setup
	<ol>
		<li>Calculate the volume of VCR working solution required to reach a final concentration of 1 &#181;M in a total volume of 2.0 mL. Vortex the stock solution before pipetting.</li>
		<li>Add AIM V medium, then 2.5 &#215; 106&#160;cells, and finally the VCR solution to a 2.0 mL microtube. Vortex briefly and spin down lightly (5 s) to collect any liquid from the tube cap.</li>
		<li>Incubate the open tubes at 37 &#176;C, 5% CO2&#160;for 3 h.</li>
		<li>After incubation, close the lids and centrifuge the samples at 300 &#215; g for 5 min at RT.</li>
		<li>Carefully discard the supernatant using a pipette, then add 1 mL of PBS at RT to the tube.</li>
		<li>Repeat the centrifugation at 300 &#215; g for 5 min at RT, supernatant removal, and PBS wash two additional times, for a total of three washes.</li>
		<li>Carefully remove all supernatant using a pipette, resuspend the pellet in 100 &#181;L of PBS, and vortex until the pellet is fully resuspended.</li>
		<li>Add 400 &#181;L of cold methanol + 0.1 &#181;M vinblastine (-20 &#176;C) directly to the sample, avoiding contact with the tube walls.</li>
		<li>Store the samples overnight at -80 &#176;C. 
		&#8203;NOTE: Samples can be stored at -80 &#176;C for a few weeks (safe stop).</li>
	</ol>
	</li>
</ol>
2. Sample processing for LC-MS/MS (Day 2)
<ol>
	<li>Sample extraction
	<ol>
		<li>Pre-cool the centrifuge to 4 &#176;C before removing samples from the -80 &#176;C freezer.</li>
		<li>Immediately after taking the samples from the freezer, centrifuge them at 13,000 &#215; g for 20 min at 4 &#176;C.</li>
		<li>Transfer 300 &#181;L of supernatant to a 1.5 mL microtube.</li>
		<li>Dry the samples completely using a vacuum concentrator at 40-45 &#176;C for 1 h and 40 min. 
		NOTE: Dried pellets can be stored at -20 &#176;C for a few weeks (safe stop).</li>
		<li>Resuspend each pellet in 400 &#181;L of water + 0.1% formic acid (previously prepared). Vortex gently until the pellet is fully dissolved.</li>
		<li>Centrifuge the samples again at 13,000 &#215; g for 20 min at 4 &#176;C.</li>
		<li>Transfer 300 &#181;L of supernatant to a 2.0 mL screw-top vial for LC-MS/MS analysis.</li>
	</ol>
	</li>
</ol>
3. LC-MS/MS setup and data acquisition
<ol>
	<li>Mobile phase preparation and column conditioning
	<ol>
		<li>Prepare Solvent A by mixing ultrapure water with 0.1% formic acid.</li>
		<li>Prepare Solvent B by mixing acetonitrile with 0.1% formic acid. 
		CAUTION: Acetonitrile is toxic and flammable; handle in a chemical fume hood.</li>
		<li>Sonicate each solvent in an ultrasonic bath for 5 min. Connect the bottles to the pump module and purge the system at high flow to remove contaminants.</li>
		<li>Install a reversed-phase BEH C18 column (1.7 &#181;m, 2.1 mm &#215; 50 mm). 
		NOTE: To perform the analysis, this study used an Acquity H-Class chromatographic system coupled to a Xevo TQS-Micro triple-quadrupole mass spectrometer.</li>
		<li>Use a binary gradient composed of Solvents A and B, both previously prepared.</li>
		<li>Run the chromatographic gradient from 5% to 95% of Solvent B over the first 2 min, followed by 2 min of Solvent A for column washing and re-equilibration.</li>
		<li>Set the flow rate to 0.2 mL/min throughout the 4-min run. Maintain the column temperature at 40 &#176;C and inject 10 &#181;L of each sample.</li>
	</ol>
	</li>
	<li>LC-MS/MS method setup
	<ol>
		<li>Open the LC-MS/MS software. Click File &#62; New &#62; MS Method to start a new method.</li>
		<li>Go to the Function tab and set the function type to multiple reaction monitoring (MRM). Enter ion transitions and parameters listed in Table 1 for VCR and VINB (internal standard).</li>
		<li>Set the Acquisition Time to 4 min. Click Apply to save. 
		NOTE: Do not exceed 4 min to ensure column re-equilibration between injections.</li>
		<li>Configure UHPLC parameters under Inlet Method &#62; Edit LC Method and save as a .mth file (Table 2).</li>
		<li>Set source parameters under the Tune tab according to Table 3. Save as a .tun file.</li>
		<li>Save the complete method: File &#62; Save As, and Export as a .mth file.</li>
	</ol>
	</li>
	<li>Creating a sample list and starting the run
	<ol>
		<li>In the software&#39;s home screen, open Sample List and create a list with the sample names, injection volumes, vial positions, and the method to be used.</li>
		<li>Place a vial with ultrapure water in the autosampler tray and designate it as a blank. Run the blank between every set of triplicate injections to avoid carryover. 
		NOTE: Before starting the run, ensure the column pressure (&#916;P) is stable and less than 10 psi.</li>
		<li>Select the desired rows in the Sample List and click Run &#62; OK to begin data acquisition.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Data visualization and export
	<ol>
		<li>After the run completes, view chromatograms by selecting a sample and clicking Chromatogram in the top menu bar.</li>
		<li>On the home screen, click on TargetLynx, select the files that just ran, and click Process &#62; Select the vincristine method &#62; OK, to generate output tables.</li>
		<li>Click Report &#62; Export Table and save the processed data as a .csv or .xlsx file. 
		NOTE: Export raw peak areas for both the analyte and internal standard.</li>
	</ol>
	</li>
	<li>Data organization and ratio calculation
	<ol>
		<li>Open the exported file and create the following columns:
		<ol>
			<li>Sample: Add the sample or vial name.</li>
			<li>Run: Indicate which replicate the value corresponds to.</li>
			<li>VCR Area: Input the area under the peak for vincristine.</li>
			<li>VINB Area: Input the area under the peak for vinblastine.</li>
			<li>VCR/VINB Ratio: Normalize each VCR value by dividing it by its corresponding VINB value.</li>
		</ol>
		</li>
		<li>Add columns for standard deviation values of VCR area, VINB area, and VCR/VINB ratio for each triplicate set. 
		&#8203;NOTE: Normalization using the internal standard allows correction for variation in extraction or injection volume.</li>
	</ol>
	</li>
	<li>Plotting results
	<ol>
		<li>Open the preferred graphing software and select the Grouped Table format.</li>
		<li>Choose Enter or import data into a new table, and under Options, select Enter 3 replicate values in side-by-side subcolumns.</li>
		<li>Plot only the normalized VCR/VINB ratios for each sample, omitting raw area values.</li>
	</ol>
	</li>
</ol>

<ol><li>Inaba, H., Pui, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+the+diagnosis+and+treatment+of+pediatric+acute+lymphoblastic+leukemia%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">Advances in the diagnosis and treatment of pediatric acute lymphoblastic leukemia.</a> J Clin Med. 10 (9), 1926(2021).</li>
<li>Pui, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Childhood+acute+lymphoblastic+leukemia%3A+progress+through+collaboration%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Childhood acute lymphoblastic leukemia: progress through collaboration.</a> J Clin Oncol. 33 (27), 2938-2948 (2015).</li>
<li>Gaudichon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+extramedullary+relapse+in+acute+lymphoblastic+leukemia%3A+reconciling+biological+concepts+and+clinical+issues%5BTitle%5D)+AND+%22Blood+Rev%22%5BJournal%5D)">Mechanisms of extramedullary relapse in acute lymphoblastic leukemia: reconciling biological concepts and clinical issues.</a> Blood Rev. 36, 40-56 (2019).</li>
<li>Dhyani, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anticancer+potential+of+alkaloids%3A+a+key+emphasis+on+colchicine%2C+vinblastine%2C+vincristine%2C+vindesine%2C+vinorelbine+and+vincamine%5BTitle%5D)+AND+%22Cancer+Cell+Int%22%5BJournal%5D)">Anticancer potential of alkaloids: a key emphasis on colchicine, vinblastine, vincristine, vindesine, vinorelbine and vincamine.</a> Cancer Cell Int. 22, 206(2022).</li>
<li>Mateus, A., Matsson, P., Artursson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+measurement+of+intracellular+unbound+drug+concentrations%5BTitle%5D)+AND+%22Mol+Pharmaceutics%22%5BJournal%5D)">Rapid measurement of intracellular unbound drug concentrations.</a> Mol Pharmaceutics. 10 (6), 2467-2478 (2013).</li>
<li>Chien, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+method+to+determine+intracellular+drug+concentrations+in+cellular+uptake+assays%3A+application+to+metformin+in+organic+cation+transporter+1-transfected+human+embryonic+kidney+293+cells%5BTitle%5D)+AND+%22Drug+Metab+Dispos%22%5BJournal%5D)">Rapid method to determine intracellular drug concentrations in cellular uptake assays: application to metformin in organic cation transporter 1-transfected human embryonic kidney 293 cells.</a> Drug Metab Dispos. 44 (3), 356-364 (2016).</li>
<li>Calcagno, A. M., Ambudkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+expression+of+drug+resistance-linked+ABC+transporters+in+cancer+cells+by+quantitative+RT-PCR%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Analysis of expression of drug resistance-linked ABC transporters in cancer cells by quantitative RT-PCR.</a> Methods Mol Biol. 637, 121-132 (2010).</li>
<li>Richter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+donor+to+the+lab%3A+a+fascinating+journey+of+primary+cell+lines%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">From donor to the lab: a fascinating journey of primary cell lines.</a> Front Cell Dev Biol. 9, 648272(2021).</li>
<li>Borgmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Childhood+ALL+blasts+retain+phenotypic+and+genotypic+characteristics+upon+long-term+serial+passage+in+NOD%2FSCID+mice%5BTitle%5D)+AND+%22Pediatr+Hematol+Oncol%22%5BJournal%5D)">Childhood ALL blasts retain phenotypic and genotypic characteristics upon long-term serial passage in NOD/SCID mice.</a> Pediatr Hematol Oncol. 17 (8), 635-650 (2000).</li>
<li>Rosati, M. M., Corrêa, J. R., de Sousa Mariano, S., Cardoso, A., Andrés Yunes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+model+of+leukemia+relapse+to+induction+chemotherapy+for+acute+lymphoblastic+leukemia%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Murine model of leukemia relapse to induction chemotherapy for acute lymphoblastic leukemia.</a> J Vis Exp. (224), e68797(2025).</li>
<li>Dams, R., Huestis, M. A., Lambert, W. E., Murphy, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+effect+in+bioanalysis+of+illicit+drugs+with+LC-MS%2FMS%3A+influence+of+ionization+type%2C+sample+preparation%2C+and+biofluid%5BTitle%5D)+AND+%22J+Am+Soc+Mass+Spectrom%22%5BJournal%5D)">Matrix effect in bioanalysis of illicit drugs with LC-MS/MS: influence of ionization type, sample preparation, and biofluid.</a> J Am Soc Mass Spectrom. 14 (11), 1290-1294 (2003).</li>
<li>Yang, Y., Cruickshank, C., Armstrong, M., Mahaffey, S., Reisdorph, R., Reisdorph, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+sample+preparation+approach+for+mass+spectrometry-based+profiling+of+plasma+results+in+improved+coverage+of+metabolome%5BTitle%5D)+AND+%22J+Chromatogr+A%22%5BJournal%5D)">New sample preparation approach for mass spectrometry-based profiling of plasma results in improved coverage of metabolome.</a> J Chromatogr A. 1300, 217-226 (2013).</li>
<li>Zhang, J., Dasgupta, A., Ayala, R., Bonapace, C., Kassim, S., Faustino, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Internal+standard+variability%3A+root+cause+investigation%2C+parallelism+for+evaluating+trackability+and+practical+considerations%5BTitle%5D)+AND+%22Bioanalysis%22%5BJournal%5D)">Internal standard variability: root cause investigation, parallelism for evaluating trackability and practical considerations.</a> Bioanalysis. 16 (15), 771-776 (2024).</li>
</ol>

The authors declare no conflicts of interest.

Accurate assessment of intracellular drug levels in primary leukemic cells is essential for understanding drug handling at the cellular level and for supporting the development of functional assays that complement molecular profiling. Existing analytical approaches often require large numbers of cells, involve lengthy processing steps, or rely on indirect readouts that do not reflect the true intracellular availability of chemotherapeutic agents5,6. These limitat...

Acute lymphoblastic leukemia (ALL) accounts for approximately 25% of all childhood cancers and remains the most common pediatric malignancy1,2. Although advances in therapy have markedly improved survival rates, now exceeding 90%-the biological complexity of ALL continues to drive efforts to develop tools that better characterize the disease at the cellular level3.
Vinca alkaloids, particularly VCR, are key components of ALL treatment protocols4. Because VCR exerts its cytotoxic effects intracellularly, understanding its uptake and accumulation in leukemic cells is fundamental for studies involving drug response, pharmacodynamics, and cellular behavior in ex vivo systems. However, most available approaches for assessing intracellular drug levels rely on indirect measurements or semi-quantitative methods, limiting reproducibility and sensitivity5,6,7.
In parallel, many investigations still depend heavily on leukemia cell lines, which lack the biological heterogeneity and clinical relevance of patient-derived primary cells. Primary samples often present challenges such as limited cell numbers, variable sample quality, and the need for highly sensitive analytical techniques capable of detecting low-abundance intracellular compounds8.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides the specificity and sensitivity required for precise quantification of chemotherapeutic agents, yet standardized protocols for measuring intracellular VCR directly in primary leukemic cells remain scarce. Establishing robust micromethods compatible with limited patient material may facilitate a broad range of applications, including pharmacological profiling, evaluation of drug-cell interactions, and comparative studies across disease subtypes or therapeutic conditions.
Here in this protocol, we describe the development and optimization of a micromethod for ex vivo quantification of intracellular VCR in primary leukemic cells using LC-MS/MS. To obtain sufficient material for method development, patient samples were transplanted into immunodeficient NSG mice to generate PDX&#160;models9. After in vivo expansion, human leukemic cells were isolated and cryopreserved10. After, they were thawed, incubated with VCR, washed, lysed, and analyzed by LC-MS/MS to determine intracellular drug levels.
This method offers several advantages: it requires a small number of cells (2.5 million), is sensitive and rapid, and is compatible with clinically relevant primary samples. Because it directly measures intracellular drug content, it provides a reliable quantitative tool for researchers investigating drug uptake, cellular responses to chemotherapeutics, or other pharmacological questions in hematologic malignancies.
Overall, this protocol expands the analytical toolkit available for studying pediatric ALL and can be readily adapted for other drugs or sample types, supporting future investigations into leukemia biology, drug behavior, and precision pharmacology.

<table><tbody><tr><td>2.0 mL screw-top vial</td><td>Flowsupply</td><td>8-2400</td><td>Used with glass inserts with spring (5 x 29 mm) from the same company (Cat. 5-29015)</td></tr><tr><td>2.0 mL screw-top vial</td><td>Flowsupply</td><td>8-2400</td><td>Used with glass inserts with spring (5 x 29 mm) from the same company (Cat. 5-29015)</td></tr><tr><td>Acetonitrile</td><td>Merck (Sigma-Aldrich)</td><td>100030</td><td></td></tr><tr><td>Acetonitrile</td><td>Merck (Sigma-Aldrich)</td><td>24010152</td><td></td></tr><tr><td>AIM V</td><td>Thermo Fisher Scientific</td><td>12055091</td><td></td></tr><tr><td>Chromatographic system Acquity UPLC H-Class</td><td>Waters Co.</td><td>https://www.waters.com/nextgen/br/pt/products/chromatography/chromatography-systems/acquity-uplc-h-class-plus-system.html?srsltid=AfmBOoo0mjNgaucBI-Exv97cEmWE96_lVkqKi2IR0IPtA-h1feG-WvtW</td><td></td></tr><tr><td>Countess 3 FL</td><td>Thermo Fisher Scientific</td><td>A49892</td><td></td></tr><tr><td>DMSO</td><td>Merck (Sigma-Aldrich)</td><td>D 8418</td><td></td></tr><tr><td>Falcon 50 mL</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>Formic acid</td><td>Merck (Sigma-Aldrich)</td><td>F0507</td><td></td></tr><tr><td>Glass bottle</td><td>Merck (Sigma-Aldrich)</td><td>41121800</td><td></td></tr><tr><td>Incubator</td><td>PHCBI</td><td>MCO-50M-PE</td><td></td></tr><tr><td>Methanol</td><td>Merck (Sigma-Aldrich)</td><td>34860</td><td></td></tr><tr><td>Microtube 1.5 mL</td><td>Eppendorf</td><td>41121702</td><td></td></tr><tr><td>Microtube 2.0 mL</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>PBS</td><td>Merck (Sigma-Aldrich)</td><td>PPB006</td><td></td></tr><tr><td>Speevac miVac</td><td>Fisher Scientific</td><td>56340</td><td></td></tr><tr><td>The software for data acquisition</td><td>Waters Co.</td><td>MassLynx</td><td></td></tr><tr><td>Triple quadrupole mass spectrometer Xevo TQ-S micro</td><td>Waters Co.</td><td>https://www.waters.com/nextgen/us/en/products/mass-spectrometry/mass-spectrometry-systems/xevo-tq-s-micro.html?srsltid=AfmBOoqqmLRNfHGqvtnwaL7xYOOEXPV3NJ9qYNH9HAwqXKXrMvLRQXSW</td><td></td></tr><tr><td>Ultrasonic Sonicator</td><td>Quimis</td><td>Q335D</td><td></td></tr><tr><td>Vinblastine 1 mg/mL</td><td>Libbs</td><td>https://www.libbs.com.br/nossos-produtos/antineoplasico-faulblastina/</td><td>Vinblastine sulfate</td></tr><tr><td>Vincristine</td><td>Cayman Chemicals</td><td>11764</td><td></td></tr></tbody></table>

development and standardization of an ex vivo micromethod for intracellular quantification of vincristine in primary all cells by lc-ms/ms

Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS

Quantifying intracellular vincristine (VCR) in primary pediatric acute lymphoblastic leukemia (ALL) cells is essential for understanding sample-specific differences in drug uptake and for supporting experimental studies using scarce patient material. Here, we present a streamlined LC-MS/MS micromethod specifically optimized for primary ALL cells obtained through patient-derived xenografts (PDX), addressing key challenges such as limited cell availability, small cell size, and the need for reproducible recovery after multiple washing steps. Time-course assays ranging from 1 to 5 h demonstrated that a 3 h incubation period yields the highest and most consistent intracellular VCR levels, with lower variability across replicates. Method optimization also established that using 2.5 &#215; 106&#160;cells per condition improves analytical robustness compared with 1 &#215; 106&#160;cells, which showed greater dispersion in repeated experiments. Additional critical refinements included the use of cold methanol to enhance protein precipitation efficiency and the incorporation of an internal standard to monitor and minimize procedural variation. The method enables sensitive and precise quantification of intracellular VCR across relevant concentration ranges, although the lowest tested dose (0.01 &#181;M) was not quantifiable after the 3 h incubation. Overall, this micromethod provides a practical, reproducible, and scalable approach for measuring intracellular VCR in primary ALL samples. Its design supports comparative analyses, validation of ex vivo models, and future methodological applications aimed at integrating intracellular drug quantification into broader pharmacological assessments.

Representative chromatographic results obtained after intracellular accumulation assays using primary ALL PDX cells incubated with VCR and VINB are shown in Figure 1. Samples were prepared using 2.5 × 106 cells per condition. Under the chromatographic conditions described, VCR was consistently retained at 2.85 min with minimal matrix effect, and peak shape was symmetrical and well-resolved. These profiles illustrate the expected chromatographic behavior of VCR and VINB when the pr...

This protocol aimed to establish and optimize an LC-MS/MS method for the relative quantification of vincristine accumulation in primary pediatric ALL cells.

Quantifying intracellular vincristine (VCR) in primary pediatric acute lymphoblastic leukemia (ALL) cells is essential for understanding sample-specific ...

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Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Targeted Metabolomics on Rare Primary Cells

This protocol describes the isolation of small extracellular vesicles from murine skeletal muscle and bone marrow using enzymatic digestion and size-exclusion chromatography, enabling reproducible recovery of vesicle-enriched fractions from structurally complex, low-yield tissues for downstream molecular analysis.

Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Learn LC-MS/MS quantification of vincristine in pediatric ALL cells. This validated method enables precise intracellular drug measurement to optimize therapeutic monitoring and improve treatment outcomes.

Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS (Video) | JoVE Journal

Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS

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