The in vitro neurosphere assay was performed to assess the numbers of neural stem cells, as previously described [8 (link)]. Briefly, the periventricular region was dissected; cells were mechanically dissociated and placed in an enzyme solution containing 1.33 mg/ml trypsin (T1005, Sigma-Aldrich), 0.76 mg/ml hyaluronidase (H6254, Sigma-Aldrich) and 0.13 mg/ml kynurenic acid (K3375, Sigma-Aldrich) in artificial cerebrospinal spinal fluid [8 (link)]. Cells were incubated for 25 min at 37 °C, followed by centrifugation for 5 min at 1500 rpm. The supernatant was removed, and samples were placed in a trypsin inhibitor solution consisting of 0.67 mg/ml ovomucoid (LS003086, Worthington, Lakewood, NJ, USA) in serum-free media (SFM) containing l-glutamine (2 mM; Invitrogen, Waltham, MA, USA) and penicillin/streptavidin (100 U/0.1 mg/ml; Invitrogen) [8 (link)]. Samples were triturated and centrifuged for 5 min at 1500 rpm, then added to SFM supplemented with epidermal growth factor (20 ng/ml; PMG8041, GIBCO, Pittsburgh, PA, USA), basic fibroblast growth factor (10 ng/ml; PHG0266, GIBCO) and heparin (2ug/ml; H3149, Sigma-Aldrich), plated at 10 cells/ul (Tabake-Coles et al. 2008) and incubated for 7 days at 37 °C and 5% CO2. The numbers of neurospheres (> 80um in diameter) were counted.
H3149
Manufactured by Merck Group
Sourced in United States
H3149 is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications. The core function of this product is to perform precise measurements and data collection tasks within controlled laboratory environments.
Automatically generated - may contain errors
Lab products found in correlation
Pmg8041, by Thermo Fisher Scientific (2 mentions)
Phg0266, by Thermo Fisher Scientific (2 mentions)
Clarity otx lysis loading buffer v2, by Phenomenex (2 mentions)
Heparin, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Af 100 18b, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Accutase, by Merck Group (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Penicillin streptavidin, by Thermo Fisher Scientific (1 mentions)
Ovomucoid, by Worthington (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Kynurenic acid, by Merck Group (1 mentions)
Porcine gelatin, by Merck Group (1 mentions)
Huvecs, by Merck Group (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Pen strep, by Merck Group (1 mentions)
20 protocols using h3149
1
In Vitro Neurosphere Assay for Neural Stem Cell Analysis
2
Heparin Concentration in Cell Culture
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We used heparin (H3149, Sigma) at concentrations of 25–100 µg/ml in culture medium (~20 U/ml of physiological activity) or fluorescently-labelled heparin (FITC-heparin, H7482, Life Technologies, Waltham, MA) at concentrations of 1–20 µg/ml in culture medium.
3
Culturing HeLa and HUVEC Cells
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HeLa cells (ATCC) were maintained in in Dulbecco's modified Eagle's medium (DMEM; Gibco, Life Technologies, Switzerland) with the addition of 10% fetal bovine serum (FBS; Sigma), 1% penicillin-streptomycin (Pen-Strep; Sigma), and 2 mM GlutaMAX (Life Technologies). Human umbilical vein endothelial cells (HUVECs; C12203; Promocell) were maintained in M199 medium (Gibco) supplemented with 30 mg/ml endothelial cell growth supplement (ECGS; 02–102; Upstate), 10 units/ml heparin (H-3149; Sigma), and 20% FBS (Sigma). Cells were cultivated on plates precoated with 1% (wt/vol) porcine gelatin (G1890; Sigma). Both HUVECs and HeLa cells were grown as monolayers at 37.0°C and 5.0% CO2. HUVECs were not used beyond passage 6.
Hoechst 33342 (Sigma) was used postfixation at a 1:10,000 dilution throughout. Cell culture-grade dimethyl sulfoxide (DMSO), used to dissolve control experimental compounds, was obtained from Sigma.
Hoechst 33342 (Sigma) was used postfixation at a 1:10,000 dilution throughout. Cell culture-grade dimethyl sulfoxide (DMSO), used to dissolve control experimental compounds, was obtained from Sigma.
4
Cell Counting and Treatment Workflow
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HEL cells were
counted using a Countess Automated Cell Counter (Invitrogen) or Countess
3 Automated Cell Counter (Invitrogen) and seeded in 24-well plates
(Techno Plastic Products 92024) at 115,000 live cells per mL and 500
μL per well in complete medium. After 3–4 h, treatments
were performed by diluting oligonucleotides or conjugates in 50 μL
of Opti-MEM (Gibco 31985047) and directly adding the diluted oligonucleotides
or conjugates to the cells. After 24 h, the cells were transferred
to clean tubes and pelleted by centrifugation. The cells were washed
with ice-cold phosphate-buffered saline (PBS; Gibco 10010015), ice-cold
0.1 mg/mL heparin (Sigma-Aldrich H3149) in PBS, and then with ice-cold
PBS. The cells were lysed in Clarity OTX Lysis-Loading Buffer v2.0
(Phenomenex AL0-8579). Cell lysates were centrifuged at 13,000 rpm
and 4 °C for 10 min, and supernatants were transferred to clean
tubes prior to storage at −80 °C. Lysates were thawed
on ice and diluted 1:75 in ultrapure water prior to analysis by CL-qPCR.
counted using a Countess Automated Cell Counter (Invitrogen) or Countess
3 Automated Cell Counter (Invitrogen) and seeded in 24-well plates
(Techno Plastic Products 92024) at 115,000 live cells per mL and 500
μL per well in complete medium. After 3–4 h, treatments
were performed by diluting oligonucleotides or conjugates in 50 μL
of Opti-MEM (Gibco 31985047) and directly adding the diluted oligonucleotides
or conjugates to the cells. After 24 h, the cells were transferred
to clean tubes and pelleted by centrifugation. The cells were washed
with ice-cold phosphate-buffered saline (PBS; Gibco 10010015), ice-cold
0.1 mg/mL heparin (Sigma-Aldrich H3149) in PBS, and then with ice-cold
PBS. The cells were lysed in Clarity OTX Lysis-Loading Buffer v2.0
(Phenomenex AL0-8579). Cell lysates were centrifuged at 13,000 rpm
and 4 °C for 10 min, and supernatants were transferred to clean
tubes prior to storage at −80 °C. Lysates were thawed
on ice and diluted 1:75 in ultrapure water prior to analysis by CL-qPCR.
5
Detailed Mesenchymal Stem Cell Culture
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BCSC1 and BCSC2 were cultured in MSC medium prepared and cultured as established in our previous study [5 (link),27 (link),28 (link)]. The MSC medium consists of (MEBM) (Lonza, CC-3151), supplemented with 1× B27 (Gibco, 17504-001, Grand Island, NY, USA), 1× amphotericin B (Gibco, 15290-026, Grand Island, NY, USA), 1× penicillin–streptomycin (Gibco, 15140-122, Grand Island, NY, USA), epidermal growth factor (20 ng/mL, PeproTech, AF-100-15, ) Rocky Hill, NJ, USA, heparin (4 μg/mL, Sigma-Aldrich, H3149, Taufkirchen, Germany), fibroblast growth factor (20 ng/mL, PeproTech, AF 100-18B, Cranbury, NJ, USA), gentamicin (35 μg/mL, Gibco, 15750-045, Paisley, UK), and rho kinase inhibitor (500 nmol/L, Calbiochem Sigma-Aldrich, 555552, Darmstadt, Germany).
6
Isolation and Cultivation of hADSC and HUVEC
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Human adipose‐derived stem cells (hADSC, PT‐5006, Clonetics, Lonza) were cultured in keratinocyte‐SFM (17005‐042, GIBCO‐Invitrogen), supplemented with 2 mmol/L N‐acetyl‐L‐cysteine (NAC, A8199, SIGMA), L‐ascorbic acid 2‐phosphate (Asc 2P, A8960, SIGMA) and 5% foetal bovine serum (16000044, GIBCO‐Invitrogen).
Human umbilical vein endothelial cells (HUVECs, BCRC No. H‐UV001) were cultured in medium 199, supplemented with 10% foetal bovine serum, 25 U/mL heparin (H‐3149, SIGMA), 30 µg/mL endothelial cell growth supplement (ECGS, 02‐102, Millipore), 2 mmol/L L‐glutamine, 1.5 g/L sodium bicarbonate and 1X penicillin/streptomycin.
For culture media collection, cell culture was limited to eight passages. 1 × 106 hADSC cells were cultured in 10 mL serum‐free media, with/without 1 μg/mL lipopolysaccharides (LPS, L3755, SIGMA), for 24 hours. The culture media (500 mL) were harvested and centrifuged at 300 g for 5 minutes to remove cells and cell debris. The supernatants were concentrated using the Amicon® Ultra‐15 (UFC900324, Merck‐Millipore) and transferred into new tubes for further use.
Human umbilical vein endothelial cells (HUVECs, BCRC No. H‐UV001) were cultured in medium 199, supplemented with 10% foetal bovine serum, 25 U/mL heparin (H‐3149, SIGMA), 30 µg/mL endothelial cell growth supplement (ECGS, 02‐102, Millipore), 2 mmol/L L‐glutamine, 1.5 g/L sodium bicarbonate and 1X penicillin/streptomycin.
For culture media collection, cell culture was limited to eight passages. 1 × 106 hADSC cells were cultured in 10 mL serum‐free media, with/without 1 μg/mL lipopolysaccharides (LPS, L3755, SIGMA), for 24 hours. The culture media (500 mL) were harvested and centrifuged at 300 g for 5 minutes to remove cells and cell debris. The supernatants were concentrated using the Amicon® Ultra‐15 (UFC900324, Merck‐Millipore) and transferred into new tubes for further use.
7
Magnesium Plates for Heparin-Gelatin Coatings
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In this research, we sourced a magnesium ingot with 99.9% purity from New Chen Te Hang Co., LTD, Kaohsiung City, Taiwan, and shaped it into plates of dimensions 20 × 20 × 1.4 mm3, which were used as the metal bases. Table 1 provides a comprehensive chemical breakdown of the magnesium. Before any tests, each sample was polished with SiC paper, having grit sizes between 600 and 1000, procured from Sanpany Co., Taipei City, Taiwan. Post-polishing, the specimens were meticulously cleaned using ethanol within an ultrasonic bath for a span of 10 min and then dried using blown air.
Heparin sodium powders were procured from Sigma-Aldrich (H3149, Grade I-A, ≥180 units/mg), St. Louis, USA, while gelatin (Gel) was acquired from Fluka Chemie, Biochemica 48723 (bloom 160), Buchs, Germany.
Heparin sodium powders were procured from Sigma-Aldrich (H3149, Grade I-A, ≥180 units/mg), St. Louis, USA, while gelatin (Gel) was acquired from Fluka Chemie, Biochemica 48723 (bloom 160), Buchs, Germany.
8
Rat Trophoblast Stem Cell Culture and Exposure Assay
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Blastocyst-derived rat trophoblast stem (TS) cells previously generated in our laboratory (Asanoma et al. 2011 (link)) were cultured in rat TS cell medium [RPMI 1640, 20% (vol/vol) fetal bovine serum (FBS; ThermoFisher), 2-mercaptoethanol (M7522; Sigma-Aldrich), sodium pyruvate (11360-070; ThermoFisher), penicillin (15140122; ThermoFisher), and streptomycin (15140122; ThermoFisher)] supplemented with 70% rat embryonic fibroblast conditioned medium prepared as previously described (Asanoma et al. 2011 (link)), fibroblast growth factor 4 ( ; 100-31; Peprotech), and heparin ( ; H3149; Sigma-Aldrich). Rat arterial endothelial cells were purchased from VEC Technologies, Inc. and maintained in MCDB-131 complete culture medium. Cells were plated in wells at confluence and treated 12 h after plating. Cells were exposed to vehicle control (i.e., DMSO) or TCDD at 10 or , concentrations known to induce CYP1A1 in vitro (Knutson and Poland 1980 (link)). The DMSO concentration in the cell cultures was 0.05%. After 24 h, cells were harvested, medium removed, and total RNA isolated.
9
Exosome Internalization Assay
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Cells were blocked for 1 h. with different receptor antagonists: Anti-β4 Integrin (10 μg/ml, ASC-8, ab77801, abcam), Cytostatin (1.4 μg/ml, 19,602, Cedarlane), and Heparin (10 μg/ml, H3149, Sigma). In parallel, exosomes were treated for 2 h. with RGD (300 nM, 14,501–1, Cedarlane), and Collagenase I (500 μg/ml, C0130, Sigma). Afterwards, cells were washed, mixed with treated exosomes and incubated for 6 h. Exosomes internalization was analyzed as described in previous section.
10
Establishment and Culture of Glioblastoma Stem Cells
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The gliomasphere (GSC21) used in this study was generated from primary GBM tumors. Briefly, the specimens were cut into small pieces, digested into single cells with Accutase (A6964, Sigma), and red blood cells were lysed by Red Blood Cell Lysis Buffer (R1010, Solarbio). Cell suspensions were then passed through a 70 μm stainless steel mesh and re-cultured in the serum-free stem cell medium. GSC21 was cultured in DMEM/F-12 medium (10565018, Gibco) containing 2% (vol/vol) B27 supplement (17504044, Gibco), epidermal growth factor (EGF, 20 ng/ml, AF-100-15, Peprotech), basic fibroblast growth factor (bFGF, 20 ng/ml, AF-100-18B, Peprotech) and heparin (2.5 μg/ml, H3149, Sigma). Only early passage GSC cells were used for the study. GSC21 were cultured at 37°C with 5% CO2.
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