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1

Mitochondrial Function Profiling of Leukemia Cells

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Oxygen consumption rate (OCR) was measured using an XFp extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA). Leukemia cells were suspended in XF Assay Medium supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. Cells (200,000 per well) were seeded in a culture plate pre-coated with Cell-Tak (Fisher Scientific). The plate was centrifuged and left to equilibrate for 60 min in a CO₂-free incubator before being transferred to the XFp extracellular flux analyzer. The Mito Stress Test was performed as follows: 1) basal respiration was measured in XF Base Medium (1 mM sodium pyruvate, 10 mM glucose, 2 mM glutamine); 2) oligomycin (1 μM) was injected to measure respiration linked to ATP production; 3) the uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM) was added to measure maximal respiration; and 4) rotenone and antimycin A (0.5 μM each) were applied together to measure non-mitochondrial respiration. Measurement of fuel dependency and fuel flexibility was performed according to the manufacturer’s Mito fuel flex test kit protocol (Agilent Technologies).

Saito Y., Sawa D., Kinoshita M., Yamada A., Kamimura S., Suekane A., Ogoh H., Matsuo H., Adachi S., Taga T., Tomizawa D., Osato M., Soga T., Morishita K, & Moritake H. (2020). EVI1 triggers metabolic reprogramming associated with leukemogenesis and increases sensitivity to L-asparaginase. Haematologica, 105(8), 2118-2129.

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2

Measuring Metabolic Profiles with Seahorse

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Metabolic rates were determined using the Seahorse XFe24 and XFe96 Flux Analyzers (Agilent Technologies) following the manufacturer’s protocol. Briefly, the microplate was coated with 22.4 μg/ml Cell-Tak (Fisher) using 200mM sodium bicarbonate. 400,000 cells were seeded per well immediately after isolation in 50μl and 80μl of unbuffered RPMI (Sigma-Aldrich) for the XF24 and XF96 analyzers, respectively. The microplate was incubated for 30 min at 37°C to allow the cells to settle into a monolayer. Unbuffered RPMI was gently added to the wells without disturbing the monolayer to bring the assay volume to 675μl and 180μl for the XFe24 and XFe96 analyzer, respectively. The basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured, in addition to rate changes upon treatment with 5μM oligomycin (Sigma-Aldrich), 1μM FCCP (Sigma-Aldrich), and 0.75μM rotenone and 1μM antimycin A (Sigma-Aldrich).

Veglia F., Tyurin V.A., Blasi M., De Leo A., Kossenkov A., Donthireddy L., Jerrick T.T., Schug Z., Basu S., Wang F., Ricciotti E., DiRusso C., Murphy M.E., Vonderheide R.H., Lieberman P.M., Mulligan C., Nam B., Hockstein N., Masters G., Guarino M., Lin C., Nefedova Y., Black P., Kagan V.E, & Gabrilovich D. (2019). Fatty acid transporter 2 reprograms neutrophils in cancer. Nature, 569(7754), 73-78.

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3

Extracellular Flux Analysis of Cellular Metabolism

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Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies). Cells were plated on Cell-Tak (Fisher) coated XF 96-well plates at 2 × 10⁵ cells per well in assay media (DMEM supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) as previously described⁵⁸. Analysis of the extracellular acidification rate and oxygen consumption rate were performed at basal level, and after subsequent injections of oligomycin (1 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM), and rotenone/antimycin mix (0.5 μM). Where included, acute injection of anti-CD3/anti-CD28 coated beads preceded the injection of oligomycin. In some experiments, 2-deoxy-D-glucose (2-DG) was injected at a final concentration of 5 mM. Following measurements, cell number was determined, averaged per condition, and the extracellular acidification and oxygen consumption rates normalized to these values. Mitochondrial ATP production was calculated by subtracting the minimum respiration rate following oligomycin injection from the final respiration rate prior to oligomycin injection.

Vardhana S.A., Hwee M.A., Berisa M., Wells D.K., Yost K.E., King B., Smith M., Herrera P.S., Chang H.Y., Satpathy A.T., van den Brink M.R., Cross J.R, & Thompson C.B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022-1033.

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4

Quantifying Tachyzoite Ingestion Dynamics

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Tachyzoite digestion was determined using an ingestion assay as previously described (12 (link)). In brief, inducible mCherry Chinese hamster ovary (CHO) cells were plated and induced with 2 μg/ml of doxycycline for 5 days. Tachyzoites were harvested from HFF cells and allowed to invade induced CHO cells for 4 h. Tachyzoites were then mechanically lysed out of host cells, purified, treated with pronase and saponin, and imaged on Cell-Tak (Fisher Scientific)-coated slides. Samples were coded at the time of initial harvesting. For each biological replicate, more than 200 tachyzoites of each genotype were analyzed for host-derived mCherry accumulation within parasites.

Kannan G., Di Cristina M., Schultz A.J., Huynh M.H., Wang F., Schultz T.L., Lunghi M., Coppens I, & Carruthers V.B. (2019). Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance. mBio, 10(4), e01324-19.

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5

Mitochondrial Respiration Profiling of T Cells

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Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFe bioanalyzer. 3x10⁵ purified T cells per well (>3 wells per sample) were spun onto Cell-Tak (Cell and Tissue Adhesive, Cat# C354240, Fisher Scientific) coated seahorse 96 well plates and preincubated in Seahorse XF OCR media (XF Base medium + 2 mM glutamine + 1 mM sodium pyruvate + 10 mM glucose) or ECAR medium (XF Base medium + 2 mM glutamine) at 37°C for a minimum of 45 mins in the absence of CO₂. OCR was measured under basal conditions, and after the addition of the following drugs: 1 μM oligomycin, 1.5 μM flurorcarbonyl cyanide phenylhydrazone (FCCP) and 0.5μM Rotenone plus Antimycin A at the indicated time point. Measurements were taken using an Extracellular Flux Analyzer (Seahorse bioscience). ECAR was measured under basal conditions, and after the addition of the following drugs: 10 mM glucose, 1.5 μM oligomycin and 50 mM 2-DG as indicated. Measurements were taken using an Extracellular Flux Analyzer (Seahorse bioscience).

Jin R., Hao J., Yi Y., Yin D., Hua Y., Li X., Bao H., Han X., Egilmez N.K., Sauter E.R, & Li B. (2021). Dietary fats high in linoleic acids impair anti-tumor T cell responses by inducing E-FABP-mediated mitochondrial dysfunction. Cancer research, 81(20), 5296-5310.

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6

Isolation and Analysis of Immune Cells

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All protocols and procedures for the collection, isolation, analysis, and storage of blood or its components have been reviewed and approved by the Institutional Review Board at the University of Alabama at Birmingham. Neutrophils and lymphocytes were isolated from whole blood of healthy individuals as previously described [36 ]. Plating of the cells following cell counting and suspension was performed in Extracellular Flux assay medium (XF-DMEM). Lymphocytes were plated at 15 × 10⁴ cells/well in the Seahorse 96 well microplate coated with Cell-Tak (CB-40242, Fisher). Neutrophils were added in co-culture at the cell densities specified. As there is some significant variation in the degree of the oxidative burst and mitochondrial function between donors, representative data from a single donor is reported for each experiment presented in this study. Key findings were verified on a minimum of 3 donors with 3–6 technical replicates per experiment. Overall, cells were obtained from 10 healthy individual donors.

Kramer P.A., Prichard L., Chacko B., Ravi S., Overton E.T., Heath S.L, & Darley-Usmar V. (2015). Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils. Clinical science (London, England : 1979), 129(6), 489-504.

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7

Seahorse XF96 Analyzer: Measuring Cellular Respiration

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OCR was measured using the Seahorse XF96 analyzer (Seahorse Bioscience,
Billerica, MD). Primary cells were suspended in XF Assay Medium supplemented
with 25 mM glucose, 1 mM pyruvate and PGF cocktail. 60,000-100,000 cells were
seeded per well of a Seahorse XF96 cell culture plate (35 μl volume)
pre-coated with Cell-tak (Fisher Scientific). Cells were left to adhere for a
minimum of 30 minutes (min) in a CO2-free incubator at 37°C, after which
140 µl of XF Assay Medium were added into each well. The plate was left
equilibrating for 10 min in the CO2-free incubator before being transferred to
the Seahorse XF96 analyzer. Measurement of OCR was done at baseline and
following sequential injections of a) oligomycin (1 µM), an ATP synthase
inhibitor b) FCCP (1.6 µM), a mitochondrial uncoupler c) antimycin A (1
µM) and rotenone (1 µM; all Sigma-Aldrich) a complex III and
complex I inhibitor respectively. This enabled us to measure the OCR coupled to
ATP production, as well as the maximal and the mitochondrial OCR respectively.
OCRs were normalized by cell number.

Kuntz E.M., Baquero P., Michie A.M., Dunn K., Tardito S., Holyoake T.L., Helgason G.V, & Gottlieb E. (2017). Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemic stem cells. Nature medicine, 23(10), 1234-1240.

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8

Extracellular Flux Analysis of Cellular Metabolism

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Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies). Cells were plated on Cell-Tak (Fisher) coated XF 96-well plates at 2 × 10⁵ cells per well in assay media (DMEM supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) as previously described⁵⁸. Analysis of the extracellular acidification rate and oxygen consumption rate were performed at basal level, and after subsequent injections of oligomycin (1 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM), and rotenone/antimycin mix (0.5 μM). Where included, acute injection of anti-CD3/anti-CD28 coated beads preceded the injection of oligomycin. In some experiments, 2-deoxy-D-glucose (2-DG) was injected at a final concentration of 5 mM. Following measurements, cell number was determined, averaged per condition, and the extracellular acidification and oxygen consumption rates normalized to these values. Mitochondrial ATP production was calculated by subtracting the minimum respiration rate following oligomycin injection from the final respiration rate prior to oligomycin injection.

Vardhana S.A., Hwee M.A., Berisa M., Wells D.K., Yost K.E., King B., Smith M., Herrera P.S., Chang H.Y., Satpathy A.T., van den Brink M.R., Cross J.R, & Thompson C.B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022-1033.

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9

Quantifying Parasite Ingestion in Host Cells

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Parasite ingestion was determined using modifications of a previously reported assay (16 (link)). In brief, mCherry was expressed in inducible Chinese hamster ovary (iCHO) cells with the addition of 2 μg/ml of doxycycline (DOX). Parasites from infected iCHO cells were harvested, purified, treated with pronase and saponin, and imaged on Cell-Tak (Fisher Scientific)-coated slides using a Zeiss Axiovert Observer Z1 inverted fluorescence microscope. For each biological replicate, more than 97 parasites of each genotype or treatment were enumerated for host-derived mCherry accumulation within parasites. Samples were coded during the time of harvesting to blind the experimenter during imaging and quantification.
In this study, we refer to the in vitro conditions that promote tachyzoite conversion to bradyzoite cysts as “bradyzoite-inducing conditions.” This includes the use of alkaline (conversion) media and growth without CO₂. For all experiments, 2 μg/ml DOX was used. Detailed changes for each ingestion assay are described below.

Kannan G., Thaprawat P., Schultz T.L, & Carruthers V.B. (2021). Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites. mSphere, 6(1), e00934-20.

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10

Seahorse XF96 Analyzer: Measuring Cellular Respiration

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OCR was measured using the Seahorse XF96 analyzer (Seahorse Bioscience,
Billerica, MD). Primary cells were suspended in XF Assay Medium supplemented
with 25 mM glucose, 1 mM pyruvate and PGF cocktail. 60,000-100,000 cells were
seeded per well of a Seahorse XF96 cell culture plate (35 μl volume)
pre-coated with Cell-tak (Fisher Scientific). Cells were left to adhere for a
minimum of 30 minutes (min) in a CO2-free incubator at 37°C, after which
140 µl of XF Assay Medium were added into each well. The plate was left
equilibrating for 10 min in the CO2-free incubator before being transferred to
the Seahorse XF96 analyzer. Measurement of OCR was done at baseline and
following sequential injections of a) oligomycin (1 µM), an ATP synthase
inhibitor b) FCCP (1.6 µM), a mitochondrial uncoupler c) antimycin A (1
µM) and rotenone (1 µM; all Sigma-Aldrich) a complex III and
complex I inhibitor respectively. This enabled us to measure the OCR coupled to
ATP production, as well as the maximal and the mitochondrial OCR respectively.
OCRs were normalized by cell number.

Kuntz E.M., Baquero P., Michie A.M., Dunn K., Tardito S., Holyoake T.L., Helgason G.V, & Gottlieb E. (2017). Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemic stem cells. Nature medicine, 23(10), 1234-1240.

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Cell tak

Lab products found in correlation

36 protocols using cell tak

Mitochondrial Function Profiling of Leukemia Cells

Measuring Metabolic Profiles with Seahorse

Extracellular Flux Analysis of Cellular Metabolism

Quantifying Tachyzoite Ingestion Dynamics

Mitochondrial Respiration Profiling of T Cells

Isolation and Analysis of Immune Cells

Seahorse XF96 Analyzer: Measuring Cellular Respiration

Extracellular Flux Analysis of Cellular Metabolism

Quantifying Parasite Ingestion in Host Cells

Seahorse XF96 Analyzer: Measuring Cellular Respiration

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