Phg0311l

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PHG0311L is a laboratory pH meter designed for precise pH measurement. It features a high-resolution LCD display and intuitive user interface. The device is capable of conducting pH, mV, and temperature measurements. Detailed specifications and intended use cases are not available.

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Lab products found in correlation

14 protocols using phg0311l

Quantification of Phosphorylated EGFR

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Quantification of phosphorylated EGFR levels was performed using the HTRF-Cellular kits: Phospho-EGFR (Tyr1068) Cellular Assay Kit (64EG1PEG, cisbio) and Total EGFR Cellular Assay Kit (64NG1PEG, cisbio). The specific EGFR phosphorylation on Tyr1068 and the total EGFR were measured after EGF-stimulation (20 ng/mL EGF (recombinant human epidermal growth factor, PHG0311L, Thermo Fisher Scientific) in serum-free DMEM/F12 medium). We followed the two-plate assay protocol provided by the vendor (SI Method S9 for a detailed description).

Liu Y.L., Chou C.K., Kim M., Vasisht R., Kuo Y.A., Ang P., Liu C., Perillo E.P., Chen Y.A., Blocher K., Horng H., Chen Y.I., Nguyen D.T., Yankeelov T.E., Hung M.C., Dunn A.K, & Yeh H.C. (2019). Assessing metastatic potential of breast cancer cells based on EGFR dynamics. Scientific Reports, 9, 3395.

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EGFR-Gapmer ASO Binding Kinetics

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Purified recombinant EGF (PHG0311L, ThermoFisher Scientific) or EGFR protein (PV3872, ThermoFisher Scientific) were incubated with FITC-labeled 2′-MOE gapmer PS-ASOs (IONIS ID 256903) or phosphodiester ASO (synthesized by Integrated DNA Technologies) in 100 μl binding buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol) as previously described (28 ). Reactions contained purified EGF at concentrations ranging from 3 nM to 3 μM or purified recombinant EGFR protein at concentrations ranging from 5 to 150 nM. After 1 h incubation at 37°C, the samples were loaded on a Hybond ECL nitrocellulose membrane (GE Healthcare) and soaked in wash buffer containing 20 mM Tris–HCl, pH 7.5 and 250 mM NaCl. The protein-bound ASOs were transferred to the membrane by applying a vacuum in a 96-well Bio-Rad Bio-Dot apparatus. After washing, the membranes were then air-dried and scanned using a PhosphorImager (GE Healthcare). The signal intensity was quantified using ImageJ, fitted as Hill Slope and the Kds were calculated using Prism.

Wang S., Allen N., Vickers T.A., Revenko A.S., Sun H., Liang X.H, & Crooke S.T. (2018). Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Research, 46(7), 3579-3594.

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Chromatin-associated RNA Profiling after EGF Stimulation

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The cells were maintained in 0.5% FBS-containing medium for 48 h and then subjected to EGF stimulation for 20 min by adding 100 ng/mL EGF (Thermo Fisher Scientific, PHG0311L). For inhibitor treatment, the cells were incubated in medium containing 1 µM vemurafenib, 200 nM PD0325901, or 1 µM SCH772984 for 3 h before collection. The chromatin-associated RNA fraction was prepared as described previously (Lai et al. 2015 (link)). To prepare the samples for ChIP, the cells were fixed with 1% formaldehyde for 10 min in a culture dish followed by incubation in 0.125 M glycine at room temperature to halt the fixation. The cells were washed, scraped, and pelleted in cold PBS. ChIP was performed as described previously (Lai et al. 2015 (link)).

Yue J., Lai F., Beckedorff F., Zhang A., Pastori C, & Shiekhattar R. (2017). Integrator orchestrates RAS/ERK1/2 signaling transcriptional programs. Genes & Development, 31(17), 1809-1820.

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Cell Culture Protocols for MCF10A, MCF7, T47D, 293T, and CHO Cells

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MCF10A/MCF10AT cells were maintained in DMEM/F12 HEPES (11330032, ThermoFisher Scientific), supplemented with following additives at the given final concentration from the indicated sources: horse serum (5%, S1215OH, Atlanta Biologicals), hydrocortisone (0.5 mg/mL, H0888, Sigma-Aldrich), cholera toxin (100 ng/mL, C8052, Sigma-Aldrich), insulin (10 µg/mL, I0516, Sigma-Aldrich), penicillin/streptomycin (1%, 15140–122, ThermoFisher Scientific), and EGF (20 ng/mL, PHG0311L, ThermoFisher Scientific). MCF7/T47D/293 T cells were maintained in DMEM (21041025, ThermoFisher Scientific), supplemented fetal bovine serum (FBS; 10%, MT35011CV, Corning) and penicillin/streptomycin (1%, 15140–122, ThermoFisher Scientific). CHO cells were grown in Ham’s F-12K (Kaighn’s) Medium (21127022, ThermoFisher Scientific), with FBS (10%, MT35011CV, Corning) and penicillin/streptomycin (1%, 15140–122, ThermoFisher Scientific). For all serum-starvation studies, DMEM/F-12, no phenol red (21041025, ThermoFisher Scientific) was supplemented with bovine serum albumin (0.1%, 10735078001, Sigma-Aldrich). All cell lines were purchased through ATCC.

Grible J.M., Zot P., Olex A.L., Hedrick S.E., Harrell J.C., Woock A.E., Idowu M.O, & Clevenger C.V. (2021). The human intermediate prolactin receptor is a mammary proto-oncogene. NPJ Breast Cancer, 7, 37.

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Wound Healing Migration Assay

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HCT116 cells were plated at 1.25×10⁶ cells in 2mL medium in a 6-well plate and allowed to adhere overnight and reach 90% confluency. Cells were scratched using a sterile 10μL pipette tip down the middle of the well to create a “wound”. Scratched cells were washed gently with PBS before being stimulated with a final concentration of 100ng/mL of EGF (Invitrogen # PHG0311L) and immediately treated with either 1.5μM 20-223 or DMSO control. Directly after the start of treatment, cells were taken to the live cell imaging facility where they were imaged every 15 minutes over a 36 hour time course (only the first 24 hours were considered for migration purposes). Migration assays were performed in triplicate (n=3).

Robb C.M., Kour S., Contreras J.I., Agarwal E., Barger C.J., Rana S., Sonawane Y., Neilsen B.K., Taylor M., Kizhake S., Thakare R.N., Chowdhury S., Wang J., Black J.D., Hollingsworth M.A., Brattain M.G, & Natarajan A. (2017). Characterization of CDK(5) inhibitor, 20-223 (aka CP668863) for colorectal cancer therapy. Oncotarget, 9(4), 5216-5232.

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Tumor Sphere Formation Assay

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EAC cell lines were cultured in DMEM/F12 with 2% B-27 serum-free supplement (17604-044; Invitrogen), 20 ng/mL epidermal growth factor (PHG0311L; Invitrogen), and 20 ng/mL basic fibroblastic growth factor (PHG0266; Invitrogen) for 14 days. Resulting tumor spheres were examined and counted under the microscope. The tumor spheres were collected for further qRT-PCR analysis to test the effect of hit-to-lead compounds on the stem-like target genes.

Diluvio G., Kelley T.T., Lahiry M., Alvarez-Trotta A., Kolb E.M., Shersher E., Astudillo L., Kovall R.A., Schürer S.C, & Capobianco A.J. (2023). A novel chemical attack on Notch-mediated transcription by targeting the NACK ATPase. Molecular Therapy Oncolytics, 28, 307-320.

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Notch Inhibition and Macrophage Co-culture in Aortic SMCs

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Human aortic SMCs (HaSMCs; CC-2571, P5-7, Lonza) were subjected to Notch inhibition through either direct treatment with DAPT or co-cultured with peritoneal macrophages which were pretreated with DAPT for 48 h. HaSMCs were grown in DMEM medium (10569-010; Gibco) containing 10% FBS, 1% penicillin-streptomycin, 4 µg/ml rH Insulin (12585-014; Fisher Scientific), 5 ng/ml recombinant human EGF (PHG0311L; Invitrogen) and 50 µg/ml ascorbic acid (A4544-25G; Sigma)^{15 (link)}. Peritoneal macrophages were obtained as described^{58 (link)}. Cell suspension was centrifuged at 4 °C at 2000 rpm for 5 min. Peritoneal macrophages were co-cultured with HaSMCs for 48 h in plain DMEM media. Culture media was processed for gelatin zymography and cells were processed for qRT-PCR analysis.

Sharma N., Dev R., Ruiz-Rosado J.D., Partida-Sanchez S., Guerau-de-Arellano M., Dhakal P., Kuivaniemi H, & Hans C.P. (2019). Pharmacological inhibition of Notch signaling regresses pre-established abdominal aortic aneurysm. Scientific Reports, 9, 13458.

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Breast Cancer Cell Line Sensitivity Screening

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We used following cell lines. CAMA-1 (ATCC® HTB-21^™) is a luminal-type human breast cancer cell line^{79 (link),80 (link)}. HCC-38 (ATCC® CRL-2314^™) is a human mammary gland-derived breast cancer cell line^{81 (link)}. MDA-MB-361 (ATCC® HTB-27^™) is a human breast adenocarcinoma-derived cancer cell line^{82 (link)}.
CAMA-1 cells were maintained in RPMI 1640 growth media (Invitrogen 11875–119) supplemented with 10% FBS (Invitrogen 16000–044), 1% Penicillin/Streptomycin (Invitrogen 15140–122), 5 μg/ml bovine insulin (Akron Biotech AK8213–0100) and 10 ng/ml human epidermal growth factor (Invitrogen PHG0311L). Cells were plated in 96-well plates at 25 × 10⁴ cells/ml and allowed to grow overnight. After 24 hours, compounds were added at 9, 3x dilutions from 100 μM final concentration in growth media. MDA-MB-361 and HCC-38 cells were maintained in growth media: DMEM/F12 (Invitrogen 11320–033) supplemented with 10% FBS (Invitrogen 16000–044) and 1% Penicillin/Streptomycin (Invitrogen 15140–122). MDA-MB-361 cells were plated in 96-well plates at 50 × 10⁴ cells/ml. HCC38 cells were plated at 25 × 10⁴ cells/ml. After 24 hours, compounds were added at 9, 3x dilutions from 100 μM final concentration in growth media.

Cavanah P., Itou J., Rusman Y., Tahara N., Williams J.M., Salomon C.E, & Kawakami Y. (2020). A nontoxic fungal natural product modulates fin regeneration in zebrafish larvae upstream of FGF-WNT developmental signaling. Developmental dynamics : an official publication of the American Association of Anatomists, 250(2), 160-174.

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Isolation of Tumor Spheres for CSC Analysis

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To obtain tumor spheres, cells were cultured in DMEM/F12 with 2% B-27 serum-free supplement (17504-044; Invitrogen), 20 ng/ml epidermal growth factor (EGF; PHG0311L; Invitrogen), and 20 ng/ml basic fibroblastic growth factor (FGF; PHG0266; Invitrogen) for 14 days to select for CSCs and early progenitor cells. Resulting tumor spheres were examined and counted under the microscope.

Wang Z., Da Silva T.G., Jin K., Han X., Ranganathan P., Zhu X., Sanchez-Mejias A., Bai F., Li B., Fei D.L., Weaver K., Carpio R.V., Moscowitz A.E., Koshenkov V.P., Sanchez L., Sparling L., Pei X.H., Franceschi D., Ribeiro A., Robbins D.J., Livingstone A.S, & Capobianco A.J. (2014). Notch Signaling Drives Stemness and Tumorigenicity of Esophageal Adenocarcinoma. Cancer research, 74(21), 6364-6374.

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Tumor Sphere Culture for CSC Enrichment

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To obtain tumor spheres, cells were cultured in DMEM/F12 with 2% B-27 serum-free supplement (17504–044; Invitrogen), 20 ng/ml epidermal growth factor (EGF; PHG0311L; Invitrogen), and 20 ng/ml basic fibroblastic growth factor (FGF; PHG0266; Invitrogen) for 14 days to select for CSCs and early progenitor cells. Resulting tumor spheres were examined and counted under the microscope.

Alvarez-Trotta A., Guerrant W., Astudillo L., Lahiry M., Diluvio G., Shersher E., Kaneku H., Robbins D.J., Orton D, & Capobianco A.J. (2021). Pharmacological disruption of the Notch1 transcriptional complex inhibits tumor growth by selectively targeting cancer stem cells. Cancer research, 81(12), 3347-3357.

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