Ht supplement

Manufactured by Thermo Fisher Scientific

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The HT supplement is a laboratory product designed to enhance the performance of certain laboratory equipment. It serves a core function of improving the overall efficiency and reliability of the equipment's operation. The specific details and intended use of this product should be obtained from the manufacturer or other authoritative sources.

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HT Supplement (50X) (2 citations)

40 protocols using ht supplement

Establishment and Maintenance of Cell Lines

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Three cell lines, CHO-hCD137, CHO-mFcγRIIb, and LLC1/OVA/hGPC3, were established previously [10] (link). CHO-hCD137, which is a CHO-DG44-based cell line transfected with an expression vector, expresses human CD137 [10] (link). CHO-mFcγRIIb is a murine FcγRII-overexpressing CHO cell line expressing murine FcγRII derived from CHO-DG44 [10] (link). LLC1/OVA/hGPC3 expresses chicken ovalbumin (OVA) and human GPC3 and was established from the murine cell line LLC1 [10] (link). CHO-mFcγRIIb and CHO-hCD137 were maintained in CHO-S-SFM II medium (Thermo Fisher Scientific) supplemented with 1% HT supplement (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific) and 500 μg/mL G418 (Thermo Fisher Scientific) in a humidified incubator maintained at 37 °C with 5% CO₂. LLC1/OVA/hGPC3 was maintained in D-MEM high glucose medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 500 µg/mL G418 (Nacalai Tesque), and 1 mg/mL Zeocin (Thermo Fisher Scientific) in a humidified incubator maintained at 37 °C with 5% CO₂.

Sugyo A., Tsuji A.B., Sudo H., Narita Y., Taniguchi K., Nemoto T., Isomura F., Awaya N., Kamata-Sakurai M, & Higashi T. (2022). In vivo validation of the switch antibody concept: SPECT/CT imaging of the anti-CD137 switch antibody Sta-MB shows high uptake in tumors but low uptake in normal organs in human CD137 knock-in mice. Translational Oncology, 23, 101481.

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Cell Culture Conditions for Cytotoxicity Assays

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SEM (55 (link)), Jurkat, CEM, MOLT-16 and Nalm-6 cells (DSMZ) were cultured in RPMI 1640 GlutaMax-I medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific) (R10+). BHK-CD16a (FcγRIIIa V158) cells were cultured in R10+ medium supplemented with 10 μmol/l methotrexate (Sigma-Aldrich) and 500 μg/ml geneticin (Thermo Fisher Scientific) as described before (53 (link), 54 (link), 56 (link)). CHO-CD32a (FcγRIIa H131) cells were cultivated in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (57 (link)). Medium was supplemented with 1% NEAA (Thermo Fisher Scientific), 1% sodiumpyruvat (Thermo Fisher Scientific) and 500 µg/ml geneticin. Chinese hamster ovary cells (CHO-S, Thermo Fisher Scientific) were cultured in serum-free CD-CHO medium (Thermo Fisher Scientific) containing 1% HT supplement (Thermo Fisher Scientific) and 2 mM GlutaMax (Thermo Fisher Scientific). After transfection CHO-S cells were cultured in CD OptiCHO medium (Thermo Fisher Scientific) containing 1% HT supplement, 2 mM GlutaMax and 0.1% Pluronic F-68 (Thermo Fisher Scientific).

Gehlert C.L., Rahmati P., Boje A.S., Winterberg D., Krohn S., Theocharis T., Cappuzzello E., Lux A., Nimmerjahn F., Ludwig R.J., Lustig M., Rösner T., Valerius T., Schewe D.M., Kellner C., Klausz K, & Peipp M. (2022). Dual Fc optimization to increase the cytotoxic activity of a CD19-targeting antibody. Frontiers in Immunology, 13, 957874.

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CHO-S Cell Culture Conditions

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CHO‐S cells were obtained from Cobra Biologics. They were grown in CD‐CHO supplemented with 8 mM L‐glutamine, 12.5 μg/mL puromycin and HT supplement (ThermoFisher Scientific) media for either 2 or 4 days. Then they were washed in phosphate‐buffered saline (PBS) and pelleted by centrifugation at 200 g.

Cole J., Hanson E.J., James D.C., Dockrell D.H, & Dickman M.J. (2019). Comparison of data‐acquisition methods for the identification and quantification of histone post‐translational modifications on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer. Rapid Communications in Mass Spectrometry, 33(10), 897-906.

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Cisplatin, Doxorubicin, and Inhibitor Treatments

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Cells were treated with 0–100 μM cisplatin (D3371, Tokyo Chemical Industry), 0–10 μM doxorubicin (040-21521, FUJIFILM Wako), 30 nM IACS-010759 (S8731, Selleckchem), 1 μM VE-821 (SML1415, Sigma-Aldrich), 1× HT supplement (11067030, Thermo Fisher Scientific), the deoxyribonucleoside mix (deoxyadenosine, thymidine, deoxyguanosine, and deoxycytidine; 10 μM each), or 0–1 mM HU (085-06653, FUJIFILM Wako) for the indicated time. As a negative control, cells were treated with vehicle alone.

Harada Y., Mizote Y., Suzuki T., Hirayama A., Ikeda S., Nishida M., Hiratsuka T., Ueda A., Imagawa Y., Maeda K., Ohkawa Y., Murai J., Freeze H.H., Miyoshi E., Higashiyama S., Udono H., Dohmae N., Tahara H, & Taniguchi N. (2023). Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells. eLife, 12, e83870.

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Comprehensive iPS-MSC-derived EV Isolation

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For EVs isolation, the cells were washed with PBS. To make sure that collected EVs only comes from iPS-MSCs, EVs were depleted from all solutions and EV collection-media used in the study by ultracentrifugation at 100 K x g in a 45Ti rotor. Serum-deficient/free medium consisting of Chinese hamster ovary medium HT Supplement, MEM Vitamin Solution, MEM Non-Essential Amino Acids solution, PBS (pH 7.4) (All from Thermofisher Scientific, USA), was added for 48 h before collection of the conditioned medium. After harvesting the conditioned medium in 50 mL falcon tubes, the medium was centrifuged at 300 × g for 10 min at 4°C in order to pellet the cells. Supernatant was centrifuged at 2,600 × g for 20 min at 4°C to remove cellular debris and dead cells. Subsequently, the supernatant was transferred to new tubes and centrifuged in a 45Ti rotor (Beckman, USA) for 6 h at 100 K × g.

Feng R., Ullah M., Chen K., Ali Q., Lin Y, & Sun Z. (2020). Stem cell-derived extracellular vesicles mitigate ageing-associated arterial stiffness and hypertension. Journal of Extracellular Vesicles, 9(1), 1783869.

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Patch-Clamp Characterization of KCNC3 Mutants

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CHO cells were grown in Iscove’s modified Dulbecco’s medium (Invitrogen), with 10% FBS, 100 units/ml pen/strep, 5% HT supplement (Thermo Fisher Scientific) in 5% CO₂ at 37°C. Seeded cells were transfected 24h later using Lipofectamine reagent (Invitrogen) with the mRuby2- or Clover-tagged KCNC3^WT or KCNC3^R423H constructs. On the recording day, patch electrodes were pulled from 1.5mm outside diameter borosilicate glass capillaries (World Precision Instruments). The resistance of a typical electrode was 2-3M for whole-cell recording when filled with intracellular solution (in mM) of 97.5 potassium gluconate, 32.5 KCl, 10 HEPES, 5 EGTA, and pH7.2, with KOH. The bath solution consisted of (in mM) 140 NaCl, 5.4 KCl, 1.3 CaCl₂, 25 HEPES, 33 glucose, and pH 7.4, with NaOH. Series resistance was 2-4M and was compensated by 80–85%. The data were acquired at 10 kHz and filtered at 5 kHz, using pClamp8 software (Molecular Devices).

Khare S., Nick J.A., Zhang Y., Galeano K., Butler B., Khoshbouei H., Rayaprolu S., Hathorn T., Ranum L.P., Smithson L., Golde T.E., Paucar M., Morse R., Raff M., Simon J., Nordenskjöld M., Wirdefeldt K., Rincon-Limas D.E., Lewis J., Kaczmarek L.K., Fernandez-Funez P., Nick H.S, & Waters M.F. (2017). A KCNC3 mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking. PLoS ONE, 12(5), e0173565.

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Investigating Ion Effects on Cell Culture

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To test the effect of various ions, Base Media was prepared with the following components: 1× essential amino acids (Thermo Fisher Scientific, Cat# 11130051), 1× MEM nonessential amino acid solution (Gibco, Cat# 11140076), 2 mM L-glutamine (Gemini Bio-products, Cat# 400106), 1× vitamins (Thermo Fisher Scientific, Cat# 11120052), 1× glucose (Thermo Fisher Scientific, Cat# A2494001), 50 mM HEPES (Lonza, Cat# 17-737E), 1 mM sodium pyruvate (Gibco, Cat# 11-360-070), 1× HT supplement (Thermo Fisher Scientific, Cat# 11067030), 2 mM CaCl₂, 1 mM MgSO₄, and 0.5% FBS. Base Media was sterilized through a 0.2 μm filter and stored at 4°C. Immediately prior to experiments, media was supplemented with salts at specified concentrations denoted in the figure legends. For isotonic control media, NaCl was added to a final concentration of 150 mM and KCl was added to 5 mM.

Kinnebrew M., Luchetti G., Sircar R., Frigui S., Viti L.V., Naito T., Beckert F., Saheki Y., Siebold C., Radhakrishnan A, & Rohatgi R. (2021). Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes. eLife, 10, e70504.

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Stable Heterologous Expression of Slack Channels in HEK-293 Cells

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Human Embryonic Kidney (HEK-293) cells that stably express Slack channels (Yang et al. 2006) were a kind gift from Dr. Fred Sigworth (Yale University, New Haven, CT). Cells were maintained at 37°C in a 5% CO₂ humidified incubator in a low sodium DMEM medium containing 10% Fetal Bovine Serum (FBS) (Invitrogen; heat-inactivated) and 1% penicillin-streptomycin (Invitrogen). In the serum starvation experiment, Slack stable HEK cells maintained in the above described medium were rinsed with Phosphate Buffered Saline (PBS) twice and kept in serum-free medium for 18–20 hours before being used. Chinese Hamster Ovary (CHO) cells were cultured at 37°C in 5% CO₂ in an IMDM medium supplemented with 10% FBS, 1% HT supplement (Life Technologies) and 1% penicillin-streptomycin. Both cell lines were plated in 35 mm dishes for all electrophysiology experiments, and in six-well culture plates for biochemical experiments.

Gururaj S., Fleites J, & Bhattacharjee A. (2015). Slack Sodium-activated Potassium Channel Membrane Expression Requires p38 Mitogen-Activated Protein Kinase Phosphorylation. Neuropharmacology, 103, 279-289.

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Transient Transfection of Slick Variants in CHO Cells

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Chinese Hamster Ovary (CHO) cells were cultured at 37 °C in 5% CO₂ in an IMDM medium supplemented with 10% FBS, 1% HT supplement (Life Technologies) and 1% penicillin-streptomycin. One day after plating at ~70% confluency, CHO cells were transiently transfected with 1 μg of either WT or mutated rSlick DNA in the expression plasmid pTRACER via lipofectamine (Invitrogen) and cultured for 48 hours. For the 1:1 WT: F240L Slick co-expression experiments, CHO cells were co-transfected with equal amounts (0.5 μg) of WT and mutated rSlick DNA. Cells were plated in 35 mm dishes for all electrophysiology and in 6-well plates for all biochemistry experiments.

Gururaj S., Palmer E.E., Sheehan G.D., Kandula T., Macintosh R., Ying K., Morris P., Tao J., Dias K.R., Zhu Y., Dinger M.E., Cowley M.J., Kirk E.P., Roscioli T., Sachdev R., Duffey M.E., Bye A, & Bhattacharjee A. (2017). A de novo mutation in the Sodium-Activated Potassium channel KCNT2 alters ion selectivity and causes epileptic encephalopathy. Cell reports, 21(4), 926-933.

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Targeted Genome Editing in Mouse Embryonic Stem Cells

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All experiments used the E14Tg2a.4 ^{40 (link)} male mouse ES cell line, which has a 36 kb X chromosome deletion that removes the first two exons of the Hprt gene. Cells were grown under feeder free conditions on gelatin coated plates and fed standard ES cell medium: Knockout DMEM (Life Technologies) containing 15% fetal bovine serum (HyClone), 2 mM L-glutamine (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), 0.05 mM 2-mercaptoethanol (Sigma), 1,000 U/mL ESGRO® LIF (Millipore), and penicillin-streptomycin. Cells were fed daily. Approximately 20 ug of linearized plasmid DNA was transfected into 1–1.5×10⁷ ES cells in 0.8 mL HEPES buffered saline (Sigma-Aldrich) using a Gene Pulser Xcell™ (Bio-Rad) set to 250 V and 500 μF. Ten such transfections were performed for each library. Correctly targeted cells were selected by the addition of Hypoxanthine-aminopterin-thymidine (HAT) Supplement (Life Technologies) to the ES cell medium for 3–10 days, beginning 24 hours after transfection. Following HAT selection, cells were fed ES cell medium containing 1× HT supplement (Life Technologies) for two days. Cells from the same library were pooled together and expanded on fresh plates prior to sorting.

Dickel D.E., Zhu Y., Nord A.S., Wylie J.N., Akiyama J.A., Afzal V., Plajzer-Frick I., Kirkpatrick A., Göttgens B., Bruneau B.G., Visel A, & Pennacchio L.A. (2014). Function-based Identification of Mammalian Enhancers Using Site-Specific Integration. Nature methods, 11(5), 566-571.

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