[3H]-estradiol 17 β-D-glucuronide (E217βG) (specific activity 37.2 Ci/mmol) and [3H]-cholecystokinin-8 (CCK-8) (specific activity 98.4 Ci/mmol) were purchased from Perkin Elmer Life Science (Waltham, MA, USA). Nicardipine was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) Unlabeled CCK-8, E217βG, dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM), Hank’s Balanced Salt Solution (HBSS), antibiotic antimycotic solution, trypsin–EDTA solution, Triton X-100, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly-L-lysine was purchased from Trevigen Inc. (Gaithersburg, MD, USA). Geneticin® and HEPES were purchased from BD Biosciences (Bedford, MA, USA). Bio-Safe II liquid scintillation mixture was purchased from Research Products International (Mt. Prospect, IL, USA).
Hepes
Manufactured by BD
Sourced in United States
HEPES is a commonly used buffer solution for maintaining a stable pH environment in various biological applications, such as cell culture and biochemical assays. It is a zwitterionic buffering agent that helps maintain a pH range of 7.0-7.8 at 37°C.
Automatically generated - may contain errors
Lab products found in correlation
Hepes, by Thermo Fisher Scientific (3 mentions)
Hanks balanced salt solution (hbss), by BD (2 mentions)
L glutamine, by BD (2 mentions)
Penicillin streptomycin, by BD (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nicardipine, by Santa Cruz Biotechnology (1 mentions)
Hank s balanced salt solution hbss, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
3h estradiol 17 β d glucuronide e217βg, by PerkinElmer (1 mentions)
3h cholecystokinin 8 cck 8, by PerkinElmer (1 mentions)
E217βg, by Merck Group (1 mentions)
Bio safe 2 liquid scintillation mixture, by Research Products International (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Geneticin, by BD (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
9 protocols using hepes
1
Receptor Binding Assay for E2 and CCK-8
2
Isolation of Ascites-Derived Cells
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Ascites (n = 18) were centrifuged at 600 g for 8 minutes. The pellet was resuspended in Hanks Balanced Salt Solution (HBSS, Gibco, Invitrogen, Basel, Switzerland) containing 25 mM Hepes (Gibco, Invitrogen, Basel, Switzerland) and 0.05 mg/ml gentamicin (Invitrogen, Basel, Switzerland) and centrifuged at 600 g for 8 minutes. The resulting pellet was resuspended in HBSS-Hepes 20 mM and filtered through a 100 μm mesh (BD Biosciences, San Jose, USA). The filtrate was resuspended with Dulbecco's Modified Eagles Medium (DMEM, Sigma Aldrich, St Louis, MO, USA) containing 10% fetal bovine serum (FBS, Biochrom AG, Oxoid AG, Basel, Switzerland) and 25 μg/ml plasmocin (InvivoGen, San Diego, CA, USA). Blood cells were eliminated by performing a Percoll (GE Healthcare, Zurich, Switzerland) gradient and centrifuged at 1200 g for 20 minutes (10%, 30%, 40%, and 70% of Percoll diluted in HBSS). Then, the cellular ring between layers 40% and 20% of Percoll was collected, diluted in DMEM and centrifuged at 600 g for 8 minutes. The pellet was resuspended and cells were counted and seeded in 3 cm dish.
3
Intracellular pH Measurement Protocol
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The pHi was measured using the Intracellular pH Detection Kit (BestBio, Shanghai, China) according to manufacturer’s instructions. Briefly, cells were washed once with Hank’s Balanced Salt Solution (HBSS) (BestBio, Shanghai, China) containing 20 mM HEPES (Solarbio, Beijing, China) and stained with 5 uM BBcellProbe in HBSS containing 20 mM HEPES for 45 min at 37 °C. After washing with HBSS containing 20 mM HEPES twice, fluorescence intensity of cells at 640 nm was analyzed by a BD FACS Aria II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A pH calibration standard curve was obtained using the nigericin as described in a previous study [69 (link)]. The pHi values were obtained based on the standard curve.
4
Cell Culture Protocols for Cancer Research
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The murine neuroblastoma cell line NXS2 (a gift from Dr. Holger N. Lode, University of Greifswald), the murine melanoma cell line B16-F10 (ATCC CRL-6475; ATCC, Manassas, VA, USA), the human embryonic retinoblast-derived 911 cell line35 (link) (Crucell, Leiden, the Netherlands), and human bone osteosarcoma (HOS) cell line (ATCC CRL-1543) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin (1% PEST), and 1 mM sodium pyruvate. The baby hamster kidney (BHK) cell line BHK-21 (ATCC CCL-10) was cultured in Glasgow minimal essential medium supplemented with 10% heat-inactivated FBS, 20 mM HEPES, and 10% tryptose phosphate broth (BD Biosciences, Franklin Lakes, NJ, USA). All cells were cultured in a humidified incubator with 5% CO2 at 37°C. All components and culture medium were from Thermo Fisher Scientific (Uppsala, Sweden).
5
Cryopreserved PBMC Sorting for Flow Cytometry
6
Assessing Lung Tissue Respiratory Capacity
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Pooled samples of D2 (2 lungs per pool/per stage) explants were processed to assess respiratory capacity (n ≥ 5/stage/condition). Lung tissue was washed in PBS and incubated, for 2 min, in respiration medium, at 37 °C. Respiration medium was composed by medium 199 (5.5 mM glucose) supplemented with 1% L-glutamine, 1% ITS (0.01 mg/mL recombinant human insulin, 0.0055 mg/mL human transferrin substantially iron-free, and 0.005 μg/mL sodium selenite) (BD Biosciences, USA) and 10 mM HEPES; pH was adjusted to 7.2–7.4, at 37 °C. To assess Oxygen Consumption Rate (OCR), a Clark-type electrode Oxytherm System (Hansatech, UK) was used [62 ]. Samples were transferred in 1 mL respiration medium to the pre-calibrated and thermostatized (with a water jacket, at 37ºC) electrode chamber. Oxygen recording protocol was performed for 15 min with an open-chamber plus 15 min with a closed-chamber; data acquisition refers to the last 5 min of closed-chamber mode. After each assay, tissue samples were retrieved, washed in PBS, and processed for protein extraction and quantification. Data was analyzed using Oxytrace Plus acquisition software (Hansatech) and normalized to the total amount of protein.
7
Cell Viability and Apoptosis Assay
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Cells were seeded in 24-well plates at 100,000 cells/ml in 0.5 ml, grown for 2 days, and treated with drug for 48 hours. After centrifugation and washing in cold PBS, cells were stained with propidium iodide (PI) (Sigma Aldrich, St. Louis, MO) and allophycocyanin-conjugated Annexin V in a binding buffer [0.1 M Hepes (pH 7.4), 0.14 M NaCl, and 25 mM CaCl2 solution] (BD Pharmingen, San Jose, CA) according to the manufacturer's protocol. Flow cytometric analysis was conducted on an Accuri C6 cytometer (BD Pharmingen, San Jose, CA) with the FCS Express software (De Novo Software, Los Angeles, CA).
8
Flow Cytometry Analysis of Extracellular Vesicles
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After resuspending EV pellets in 60 μl DPBS, we added saturating concentrations of labeled Abs, annexin V, and normal mouse IgG, and incubated the tubes in the dark without stirring for 15–30 min at room temperature. Various Ab concentrations at the time of staining are listed in Supplemental Materials and Methods . We diluted this Ab‐stained solution into 250 μl annexin V binding buffer (10 mM HEPES, 0.14 mM NaCl, 2.5 mM CaCl2, pH 7.4; BD Biosciences), which was then subjected to flow cytometry analysis. The DPBS and annexin V binding buffer were filtered through 0.1‐μm filters (Millipore). Flow cytometry was carried out using a FACSVerse flow cytometer (BD Biosciences). In our previous report, we implemented a method to measure particles smaller than 1 μm in size using polystyrene beads for verification and aggregating them into an observed image using side scatter.13 The flow cytometer was equipped with 405 nm, 488 nm, and 638 nm lasers to detect up to 13 fluorescent parameters. The flow rate was 12 μl/min. Forward scatter voltage was set to 381, side scatter voltage was set to 340, and each threshold was set to 200. Details of excitation and emission wavelengths as well as voltages are described in the Supplementary Materials and Methods . Flow cytometry was carried out using FACSuite software (BD Biosciences) and data were analyzed using FlowJo software.
9
CD8+ T Cell Isolation and Sorting
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Spleens from mice in the same experimental group (i.e. d8 Arm, etc.) were processed together, five at a time. Spleens were homogenized using a Miltenyi gentleMACS™ Dissociator in C tubes. CD8 T cells were enriched using an EasySep magnetic negative selection kit (Stem Cell Technologies #19853) as per the manufacturer's recommendations. Cells were washed with 1× PBS and stained with an amine-reactive dye (Biolegend # 423106) for 20 minutes at room temperature to assess cell viability, followed by an antibody cocktail in complete RPMI (cR10, RPMI-1640 media supplemented with 10% FBS, 1x non-essential amino acids (Gibco #11140050), and 10mM Hepes (Gibco #15630080), 2mM L-glutamine (Gibco #25030081), 100U/mL penicillin/streptomycin (Gibco #15140122), 14.3 uM beta-mercaptoethanol) for 45 minutes on ice. Samples were sorted on a BD FACSAria II machine into RPMI-1640 media supplemented with 50% FBS, 1% Hepes, 1% L-glutamine, 1% penicillin/streptomycin (cR50). Cells gated as live single CD8+ P14 cells designated by congenic markers. A small aliquot of all sorted samples was run as a purity check. Voltages on the machine were standardized using fluorescent targets and Spherotech rainbow beads (#URCP-50-2F).
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