Lsgs kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The LSGS Kit is a laboratory equipment product designed for Liquid Scintillation Counting (LSC) analysis. It provides the necessary components to prepare and measure radioactive samples using a Liquid Scintillation Counting system. The kit includes vials, scintillation cocktail, and other required accessories for conducting LSC experiments.

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10 protocols using lsgs kit

Overexpression of miRNA in MCF7 Cell Line

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Human breast cancer cell line MCF7 was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Stable miRNA overexpressing MCF7-miR526b and MCF7-miR655 cell lines were established as previously described [9 (link),10 (link)]. MCF7, MCF7-miR526b, and MCF7-miR655 cells were all grown in minimal essential medium (MEM) (Life Technologies, Thermofisher, Ottawa, ON, Canada) supplemented with 10% fetal bovine serum (FBS) and 1% Penstrep. Furthermore, MCF7-miR526b and MCF7-miR655 cell lines were sustained with Geneticin (Life Technologies Thermofisher, Ottawa, ON, Canada) at 200 ng/mL.
HUVECs were purchased from Life Technologies and grown in Medium 200 (GIBCO, ON) supplemented with low serum growth supplement (LSGS) kit (GIBCO, Toronto, ON, Canada) containing 2% FBS, hydrocortisone (1 µg/mL), human epidermal growth factor (10 ng/mL), basic fibroblast growth factor (3 ng/mL), and heparin (10 µg/mL). All cell lines were maintained in a humidified incubator at 37 °C with 5% CO₂.

Hunter S., Nault B., Ugwuagbo K.C., Maiti S, & Majumder M. (2019). Mir526b and Mir655 Promote Tumour Associated Angiogenesis and Lymphangiogenesis in Breast Cancer. Cancers, 11(7), 938.

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Cell Culture Protocols for Cancer Research

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The studies were conducted on human lung adenocarcinoma A549 (CCL-185; ATCC, Manassas, VA, USA) cell line, breast cancer cell line HCC1937 (CRL-2336, ATCC, Manassas, VA, USA) and NALM-6 ALL cells (CRL-3273, ATCC, Manassas, VA, USA). The cell lines were maintained in DMEM media (ThermoFisher Scientific, Waltham, MA, USA) (for A549) or RPMI 1640 media (HTC1937 and NALM6) supplemented with 10% (v/v) HI fetal bovine serum (FBS) and 100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C in 5% CO₂ atmosphere. Cell culture media and supplements were purchased from Lonza (Basel, Switzerland). In addition, human umbilical vein endothelial cells (HUVEC) were also used for the study. These cells were purchased from Gibco (Cascade Biologics, Portland, OR, USA) catalog number C0035C) and cultured in medium 200 (Gibco, catalog number M-200–500), supplemented with low serum G growth supplement kit (LSGS Kit; Gibco, catalog number S003K) at 37 °C and 5% CO₂ in an incubator (Galaxy^® R-CO2 Incubator, New Brunswick Scientific, New Brunswick, NJ, USA) in a humidified atmosphere.

Kowalczyk T., Merecz-Sadowska A., Rijo P., Isca V.M., Picot L., Wielanek M., Śliwiński T, & Sitarek P. (2021). Preliminary Phytochemical Analysis and Evaluation of the Biological Activity of Leonotis nepetifolia (L.) R. Br Transformed Roots Extracts Obtained through Rhizobium rhizogenes-Mediated Transformation. Cells, 10(5), 1242.

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Culturing Human Umbilical Vein Endothelial Cells

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All experiments were performed on human umbilical vein endothelial cells (HUVECs). These cells were purchased from Gibco (Cascade Biologics®, catalog number C0035C) and cultured in Medium 200 (Gibco, catalog number M-200-500) supplemented with Low Serum G Growth Supplement Kit (LSGS Kit; Gibco, catalog number S003K) at 37°C and 5% CO₂ in an incubator (Galaxy® 170 R-CO₂ Incubator, New Brunswick Scientific) under a humidified atmosphere.

Kowalczyk T., Sitarek P., Skała E., Rijo P., Andrade J.M., Synowiec E., Szemraj J., Krajewska U, & Śliwiński T. (2019). An Evaluation of the DNA-Protective Effects of Extracts from Menyanthes trifoliata L. Plants Derived from In Vitro Culture Associated with Redox Balance and Other Biological Activities. Oxidative Medicine and Cellular Longevity, 2019, 9165784.

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Endothelial Cell Spiking Experiments

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The endothelial cell lines HUVEC (human umbilical vein endothelial cells), L‐HMVEC (lung human microvascular endothelial cells), HPAEC (human pulmonary arterial endothelial cells), and HAEC were obtained from Lonza (Basel, Switzerland). HUVEC, HPAEC, and HAEC were cultured in Medium200 supplemented with LSGS kit (Life Technologies), whereas Lung–HMVEC (L‐HMVEC) with endothelial cell basal medium (EGM‐2; Lonza) containing EGM‐2 MV Bulletkit supplements (Lonza). Cell were maintained in culture for no more than six culture passages. For spiking experiments cells were harvested using Accutase (Sigma‐Aldrich, St.Luis, MO, USA), and resuspended in phosphate‐buffered saline without Ca²⁺Mg²⁺. After determining the cell concentration in a hemocytometer, the cell suspension was added to blood samples to achieve the theoretical cell concentrations of 100, 1000, and 10 000 cells mL⁻¹ blood. To test the Transfix collection tubes blood samples were collected in EDTA‐tubes, mixed with the HUVEC or HMVEC and then added of Transfix solution at ratio 1:5.

Farinacci M., Krahn T., Dinh W., Volk H., Düngen H., Wagner J., Konen T, & von Ahsen O. (2018). Circulating endothelial cells as biomarker for cardiovascular diseases. Research and Practice in Thrombosis and Haemostasis, 3(1), 49-58.

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Culturing U-87 Glioblastoma and HUVEC Cells

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U-87 human primary glioblastoma cells were grown in DMEM (10% FBS, 1% l-Glu, 1% penicillin streptomycin). Human umbilical vein endothelial cells (HUVECs) from Thermo Fisher Scientific were grown in Medium 200 supplemented with LSGS kit (Life Technologies). Cell culture was always performed at 37 °C in 5% CO₂ and 95% relative humidity (RH).

De Capua A., Palladino A., Chino M., Attanasio C., Lombardi A., Vecchione R, & Netti P.A. (2021). Active targeting of cancer cells by CD44 binding peptide-functionalized oil core-based nanocapsules. RSC Advances, 11(40), 24487-24499.

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Cultivation and Stimulation of Human Endothelial Cells

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Pooled primary HUVEC cells from male and female donors were purchased from Thermo Fisher Scientific (C01510C), passaged twice and cryopreserved. Early-passage cells were used for all in vitro experiments (2–4 passages total). HUVEC and E4-HUVEC cells were cultured in M200 medium (ThermoFisher Scientific M200500) supplemented with 2% FBS, Hydrocortisone 1 μg/mL, human EGF 10 ng/mL, b-FGF 3ngmL and Heparin 10 μg/mL (LSGS kit, ThermoFisher Scientific). Cells were serum-starved in M200 alone for 8h prior to treatment with rhCXCL8 (RND Systems). 293T cells were cultured in high-glucose Dulbecco’s Modified Eagles Medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 1mM sodium pyruvate and penicillin/streptomycin. Cells were cultured in a humidified 5% C0₂ atmosphere at 37C.

Binder V., Li W., Faisal M., Oyman K., Calkins D.L., Shaffer J., Teets E.M., Sher S., Magnotte A., Belardo A., Deruelle W., Gregory T.C., Orwick S., Hagedorn E.J., Perlin J.R., Avagyan S., Lichtig A., Barrett F., Ammerman M., Yang S., Zhou Y., Carson W.E., Shive H.R., Blachly J.S., Lapalombella R., Zon L.I, & Blaser B.W. (2023). Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C. Cell reports, 42(5), 112528.

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Endothelial Cell Response to SARS-CoV-2 S1 Protein

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HPAECs were grown to 80–90% confluency in complete media before any treatments. Cells were then serum starved for 12 h in FBS-free medium composed of Medium 200 supplemented with all other components of LSGS Kit except FBS (Thermo Fisher Scientific, Waltham, MA, USA). LPS-free recombinant SARS-CoV-2 Spike Protein S1 (aa 1–681, catalog no. AGX818) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were exposed to either S1 protein alone or with HbA (65 µM) for up to 24 h in serum-free growth media. After the incubation, cells were washed with pre-warmed phosphate-buffered saline (PBS) three times to remove any residual S1 or Hb proteins in the media. Cells were then lysed with RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor for further studies.

Jana S., Heaven M.R, & Alayash A.I. (2021). Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells. International Journal of Molecular Sciences, 22(16), 9041.

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Hb-Induced Oxidative Stress Response

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For all experiments HPAEC were grown to 80–90% confluency in complete media. Before any treatment, cells were serum starved for 6 h in a medium composed of Medium 200 supplemented with all the components of LSGS Kit except FBS (Thermo Fisher Scientific, Waltham, MA). The cells were then exposed to ferrous (HbFe²⁺), ferric (HbFe³⁺) or ferryl (HbFe⁴⁺) for varying periods of time up to 12 h. Antioxidants, scavengers or other protective agents were added to the media prior to the addition of Hb. In most of the experiments HPAEC were washed three times with either pre-warmed phosphate buffered saline (PBS) or with complete media to remove Hb proteins and other additions otherwise mentioned. Following exposure to specified time periods, cell lysates were prepared for further studies.

Jana S., Meng F., Hirsch R.E., Friedman J.M, & Alayash A.I. (2017). Oxidized Mutant Human Hemoglobins S and E Induce Oxidative Stress and Bioenergetic Dysfunction in Human Pulmonary Endothelial Cells. Frontiers in Physiology, 8, 1082.

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Culturing Common Cell Lines for Research

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HEK293, HUVEC, THP-1, and wild-type primary dermal fibroblast cells were purchased from American Type Tissue Collection (ATCC; Manassas, VA, USA). Primary Dermal Fibroblasts cells were grown in Media 106 with the addition of LSGS kit (S-003-10) (ThermoFisher, Rockford, IL, USA) and used between passage 4-10. HEK293 cells were maintained in 5% FBS with Improved Minimum Essential Medium (IMEM) (ThermoFisher, Rockford, IL, USA), THP-1 cells were grown in RPMI (ThermoFisher, Rockford, IL, USA) with 10% FBS following the manufacturer’s recommendation. HUVEC cells were grown in vascular cell basal medium with the addition of VEGF endothelial cell growth kit (ATCC; Manassas, VA, USA), and used between passages 3 and 8.

Ivanova M.M., Dao J., Kasaci N., Adewale B., Fikry J, & Goker-Alpan O. (2020). Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy. Biomolecules, 10(6), 837.

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Hb Variants Exposure on HPAECs

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HPAECs were grown to 80–90% confluency in complete media, before any treatments. Cells were then serum starved for 12 h in an FBS-free medium composed of Medium 200, supplemented with all other components of the LSGS Kit except FBS (Thermo Fisher Scientific, Waltham, MA, USA). The cells were exposed to various Hb variants at equimolar concentrations (100 µM) in their ferrous form (HbFe²⁺) for 24 h in an FBS-free growth media. After incubation, the cells were washed with pre-warmed phosphate buffered saline (PBS) for three times, to remove any residual Hb proteins in the media. Cells were then lysed with RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing the protease inhibitor for further studies.

Jana S., Strader M.B, & Alayash A.I. (2020). The Providence Mutation (βK82D) in Human Hemoglobin Substantially Reduces βCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells. International Journal of Molecular Sciences, 21(24), 9453.

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