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Lk003182

Manufactured by Worthington
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The LK003182 is a laboratory instrument designed for general scientific applications. It is a compact and versatile device capable of performing various laboratory tasks. The core function of this equipment is to facilitate standard laboratory procedures and measurements. No further details or interpretation about its intended use are provided.

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Lab products found in correlation

Lsoo3126, by Worthington (2 mentions) D4627, by Merck Group (2 mentions) P0781, by Merck Group (1 mentions) Lk003176, by Worthington (1 mentions) Lk003170, by Worthington (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Rpmi 1640, by Merck Group (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Dnase, by Worthington (1 mentions) P4707, by Merck Group (1 mentions) Lk003178, by Worthington (1 mentions) Lk003172, by Worthington (1 mentions) Hanks balanced salt solution (hbss), by Merck Group (1 mentions) Neurobasal, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Papain, by Worthington (1 mentions) P4937, by Merck Group (1 mentions)

4 protocols using lk003182

1

Isolation and Culture of Primary GBM Cells

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Tumor tissues were dissected from patients, ≥18 years of age with primary GBM tumors, during surgery in the Neurosurgery Department of The Second Affiliated Hospital of Anhui Medical University (Hefei, China) and collected in sterile Hibernation media and transported to the laboratory on ice within 1 hr. Patient-derived tumor tissue was cut into small pieces with a scalpel and digested for 30 min at 37°C enzymatically in a mixture consisting of Papain (20 µ/mL, #LK003176, Worthington) and DNase (2000 µ/mL, #LK003170, Worthington). Ovomucoid inhibitor (10 mg/mL, #LK003182, Worthington) was used to stop the enzymatic activity at room temperature and an erythrocyte lysis was performed for further 20 min at room temperature. Seed the primary GBM cells into a GelTrex or Matrigel-coated plastic flask and cultivate in RPMI 1640 (R8758, Sigma) supplemented with 10% FCS (F7524, Sigma) and 1% penicillin/streptomycin (P0781, Sigma). Two primary cell lines (T76, H34) isolated from patient tumor tissue were included in this experimental study. This study was approved by the Research Ethics Committee of The Second Affiliated Hospital of Anhui Medical University. Informed consent was obtained from all the patients.
Yang Z., Bian E., Xu Y., Ji X., Tang F., Ma C., Wang H, & Zhao B. (2020). Meg3 Induces EMT and Invasion of Glioma Cells via Autophagy. OncoTargets and therapy, 13, 989-1000.
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2

Primary Culture of Cortical Neurons and Astrocytes

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Mixed cultures of cortical neurons and astrocytes were obtained by dissecting brains from P0-P1 mice. Cortices from each brain were isolated, kept in HBSS (H6648, Sigma) on ice separated from each other and genotyping was performed on liver extracted from each pup.
Brain tissue was incubated in EBSS (E2888, Sigma) and papain (LK003178, Worthington Biochemical Corp.) for 40 min at 37 °C and it was then dissociated by trituration in EBSS supplemented with DNAse (LK003172, Worthington Biochemical Corp.) and papain inhibitor (LK003182, Worthington Biochemical Corp.). After spinning, the cell pellet was resuspended in Neurobasal (21103-049, Life Technologies), supplemented with B27 (17504-044, Life Technologies), Glutamax (35050-038, Life Technologies), and 100 U/ml Penicillin–Streptomycin (1514-122, Life Technologies) counted and plated to appropriate densities on coverslips (0.5·106 cells), 6-well plates (106 cells) or 96-well plates (30000 cells), coated with polylysine (P4707, Sigma). Half media changes were done every 4 or 7 days. Cultures were maintained at 37 °C and 5% CO2 in humidified atmosphere and used between 10 and 15 days in vitro unless stated differently.
Plotegher N., Perocheau D., Ferrazza R., Massaro G., Bhosale G., Zambon F., Rahim A.A., Guella G., Waddington S.N., Szabadkai G, & Duchen M.R. (2019). Impaired cellular bioenergetics caused by GBA1 depletion sensitizes neurons to calcium overload. Cell Death and Differentiation, 27(5), 1588-1603.
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3

Establishing Patient-Derived Cell Lines

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The RCMB32 and ICb-984 cell lines were generated in the laboratories of R.J.W.-R. and X.-N.L., respectively. The BT084 cell line was provided by T. Milde (Heidelberg University Hospital). The primary tumor sample (ST01) (Fig. 5, B to D) obtained from the operating room was minced and then placed in a digestion buffer consisting of 1× HBSS (14185, Thermo Fisher Scientific), papain (10 U/ml; LSOO3126, Worthington Biochemical), and DNase (250 U/ml; D4627, Sigma). Papain solution was aspirated and replaced with 1× HBSS containing ovomucoid (8 mg/ml; LK003182, Worthington Biochemical), BSA (8 mg/ml; Sigma), and DNase (250 U/ml) and then triturated using a Pasteur pipette to obtain a single-cell suspension. Cells were pelleted and resuspended in 0.02% HBSS-BSA. Cells were passed through a 70-μm nylon cell strainer (21008–952, VWR International). Of 20 tumors implanted, 5 grew to a sufficient size to resect. Cell viability and cell cycle stage were assayed by high-throughput single-cell immunofluorescence imaging, as described above; 12 individual cultures were assessed at each dose. Gli1 transcript expression was assessed by qRT-PCR, as described above, and three cultures were assessed at each dose. Final pathological report is provided in table S4.
Purzner T., Purzner J., Buckstaff T., Cozza G., Gholamin S., Rusert J.M., Hartl T.A., Sanders J., Conley N., Ge X., Langan M., Ramaswamy V., Ellis L., Litzenburger U., Bolin S., Theruvath J., Nitta R., Qi L., Li X.N., Li G., Taylor M.D., Wechsler-Reya R.J., Pinna L.A., Cho Y.J., Fuller M.T., Elias J.E, & Scott M.P. (2018). Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma. Science signaling, 11(547), eaau5147.
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4

Isolation and Purification of Granule Neuronal Precursors

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Wild-type CD1-Elite mice (strain 482, Charles River) were sacrificed at P1, P7, and P14. Thirty-five to 40 mice were used per P1 and P14 biological replicate, and 14 to 17 mice were used per P7 biological replicate. Cerebella were dissected, and GNPs were isolated and purified using a Percoll gradient, as previously described (58 ,59 ). This approach results in a population that is 95 to 99% GNPs (58 ). Briefly, cerebella were minced and then placed in a digestion buffer consisting of 1× Hanks’ balanced salt solution (HBSS) (14185, Thermo Fisher Scientific), papain (10 U/ml; LSOO3126, Worthington Biochemical), and deoxyribonuclease (DNase) (250 U/ml; D4627, Sigma). Papain solution was aspirated and replaced with 1× HBSS containing ovomucoid (8 mg/ml; LK003182, Worthington Biochemical), bovine serum albumin (BSA) (8 mg/ml; Sigma), and DNase (250 U/ml) and was then triturated using a Pasteur pipette to obtain a single-cell suspension. Cells were passed through a 70-μm nylon cell strainer (21008–952, VWR International) and purified using a step gradient of 35 and 65% Percoll (P4937, Sigma). Pellets were flash-frozen in liquid nitrogen and kept at −80°C until all samples were collected for MS.
Purzner T., Purzner J., Buckstaff T., Cozza G., Gholamin S., Rusert J.M., Hartl T.A., Sanders J., Conley N., Ge X., Langan M., Ramaswamy V., Ellis L., Litzenburger U., Bolin S., Theruvath J., Nitta R., Qi L., Li X.N., Li G., Taylor M.D., Wechsler-Reya R.J., Pinna L.A., Cho Y.J., Fuller M.T., Elias J.E, & Scott M.P. (2018). Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma. Science signaling, 11(547), eaau5147.
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