Vincristine (purity >98%) and vinblastine (purity ≥95%; internal standard (IS)) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). We initially made sure that none of the patients were treated with vinblastine. So, it was selected as the internal standard due to its structural similarity to vincristine. Stock standard solution of vincristine was prepared in Methanol (5.0 mL; 1000 mg L−1), and kept at a −20 °C. Distilled water (6 times distilled) was purchased from Shaid Ghazi Pharmaceutical Company of Tabriz (Tabriz, Iran). HBA reagent (MTOAC; purity >97%) were obtained from Aladdin Biochemical Company (Shanghai, China). Methanol, acetonitrile, NaCl, KHCO3, phosphate salt and HBD reagents were purchased from Merck Company (Darmstadt, Germany).
Khco3
Manufactured by Merck Group
Sourced in Germany, United States
KHCO3 is a chemical compound with the formula KHCO3. It is a white, crystalline solid that is soluble in water. KHCO3 is commonly used as a laboratory reagent and has various applications in chemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Nh4cl, by Merck Group (32 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (14 mentions)
Facsuite software, by BD (6 mentions)
Ethylene diamine tetraacetic acid (edta), by AppliChem (6 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
K2hpo4, by Merck Group (4 mentions)
Methanol, by Merck Group (3 mentions)
Sodium pyruvate, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Merck Group (3 mentions)
Non essential amino acids (neaa), by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
Asparagine, by Merck Group (3 mentions)
β mercaptoethanol, by Merck Group (3 mentions)
Dynabeads b220 depletion kit, by Thermo Fisher Scientific (3 mentions)
Anti cd3 500a2, by Thermo Fisher Scientific (3 mentions)
Anti cd28 37, by Thermo Fisher Scientific (3 mentions)
Htec 500 system, by Eicom (3 mentions)
Edta 2na, by BD (3 mentions)
Sodium decane 1 sulfonate, by Thermo Fisher Scientific (3 mentions)
Rotisolv hplc gradient grade water, by Merck Group (3 mentions)
69 protocols using khco3
1
Quantitative Analysis of Vincristine
2
Neutrophil Isolation and EV Production
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Blood was drawn with a Vacutainer Eclipse needle into a Vacuette sodium heparine 9 ml tube (Greiner Bio-One, Alphen aan de Rijn, Netherlands). Blood was diluted with HBSS (Sigma Aldrich H6136) after which granulocytes and erythrocytes were separated from PBMC by density gradient centrifugation on 20 ml Lymphoprep (STEMcell Technologies, Köln, Germany). The granulocyte/erythrocyte pellet was subsequently transferred to a new 50 mL tube followed by erythrocyte lysis in ice-cold erythrocyte lysis buffer (0.155 M NH4 Cl (Sigma), 1 mM KHCO3 (Merck), 80 µM EDTA (Merck) in Milli-Q) in 2 consecutive rounds. The final cell pellet was washed in PBS, centrifuged 8 min at 400 g and seeded in IMDM + ultraglutamine (Gibco) supplemented with 1% EV-depleted FCS (Sigma-Aldrich, F7524), and 86 µg/mL gentamycin (Duchefa, DUC586714) at 1E6 cells/mL for EV production. Purity of isolated cells was determined on flow cytometry (see below). Neutrophils were positive for CD15 and contained less than 3% PBMC (determined by FCS/SSC detection). Where indicated, cells were stimulated with 100 ng/mL LPS (O111-B4, cat L2360, Sigma-Aldrich, St. Louis, MO). EV were produced in 2 h with 9x10E6 neutrophils, after which medium was collected for EV isolation. Cell viability after culture was checked by PI or DAPI staining, which was >90% for all cultures.
3
CO2 Electroreduction in Electrochemical Cell
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CO2 electroreduction was performed in a custom built three-electrode electrochemical cell45 (link). The reference and counter electrodes were, respectively, a Ag/AgCl (saturated KCl, Pine) and a coiled Pt wire. The cathodic and anodic compartments were separated by an anion exchange membrane (Asahi Glass). Both compartments were filled with CO2-saturated 0.1 M KHCO3 (99.7%, Merck). During 60 min chronoamperometry, 20 cm3 min−1 of CO2 was continuously flowed into the electrolyte. The gas outlet of cathodic compartment was connected to a gas chromatograph (GC, Agilent 7890A) for the online quantification of gas products. The liquid products were quantified by headspace gas chromatography (HS-GC, Agilent 7890B) and high performance liquid chromatography (HPLC, Agilent 1260) after electrolysis. The voltage drop was automatically compensated using the current-interrupt mode available in the potentiostat (Gamry 600). The voltage was converted to the RHE scale and the current density was normalized to the exposed geometric surface area. All the detected products were quantified in terms of their FE. The FE of product X is defined as: Each reported FE value was the average of three independent sets of measurements.
4
Isolation and Expansion of Lymphocytes
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Lymph nodes were passed through a 30-μm cell strainer (Sysmex, #04-004-2326) and spleens were passed through a 70-μm cell strainer (Greiner Bio-One, #542070), followed by gravity centrifugation (400 rcf, 4 °C, 10 min). Spleen samples underwent erythrocyte lysis with BD Pharm Lyse (BD Biosciences, #555899) or ACK solution (NH4Cl 8.024 mg/l (Merck, #1011451000), KHCO3 1.001 mg/l (Merck, #1048540500), EDTA.Na2·2H2O 3.722 mg/l (Merck, #1084181000)).
In retroviral transduction experiments, B cells were depleted from the cell suspensions using the Dynabeads B220 depletion kit (Thermo Fisher, #11441D). The remaining T cells were cultured in the presence of 0.5 μg/ml anti-CD3 (500A2, Invitrogen, #16-0033-86) and anti-CD28 (37.51, Invitrogen, #16-0281-86) stimulating antibodies in complete DMEM (Sigma-Aldrich, #D5671) (supplemented with 2 mM L-Glutamine (Sigma-Aldrich, #G7513), 100 μg/ml Penicillin/Streptomycin (Sigma-Aldrich, #4333), 36 mg/l Asparagine (Sigma-Aldrich, #A4284), 1 mM Sodium-Pyruvate (Sigma-Aldrich, #8636), 10 ml/l Non-essential amino acids (Sigma-Aldrich, #M7145), 4 μl/l β-Mercaptoethanol (Merck KGaA, #805740) and 10% fetal calf serum (Sigma-Aldrich, #S0615)).
In retroviral transduction experiments, B cells were depleted from the cell suspensions using the Dynabeads B220 depletion kit (Thermo Fisher, #11441D). The remaining T cells were cultured in the presence of 0.5 μg/ml anti-CD3 (500A2, Invitrogen, #16-0033-86) and anti-CD28 (37.51, Invitrogen, #16-0281-86) stimulating antibodies in complete DMEM (Sigma-Aldrich, #D5671) (supplemented with 2 mM L-Glutamine (Sigma-Aldrich, #G7513), 100 μg/ml Penicillin/Streptomycin (Sigma-Aldrich, #4333), 36 mg/l Asparagine (Sigma-Aldrich, #A4284), 1 mM Sodium-Pyruvate (Sigma-Aldrich, #8636), 10 ml/l Non-essential amino acids (Sigma-Aldrich, #M7145), 4 μl/l β-Mercaptoethanol (Merck KGaA, #805740) and 10% fetal calf serum (Sigma-Aldrich, #S0615)).
5
Isolation and Cultivation of Adipocyte Precursor Cells
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Subcutaneous AT samples were obtained from children undergoing bariatric or elective orthopedic surgery (Supplementary Table 1 ). SVF cells and adipocytes were isolated and preserved for later RNA isolation as previously described19 (link),46 . The remaining SVF cells were filtered through a nylon mesh with 30-µm pore size. Erythrocytes were removed with erythrocyte lysis buffer (0.154 M NH4Cl (Sigma-Aldrich), 0.01 M KHCO3 (Merck) and 0.1 mM EDTA (Sigma-Aldrich)). SVF cells were frozen in liquid nitrogen in Dulbecco’s modified Eagle medium/Ham F-12 culture medium (DMEM/F-12) (Life Technologies) containing 10% fetal bovine serum (FBS) (Biochrom) and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Cells were thawed and 24 h after seeding cells were washed three times with phosphate-buffered saline to select for adipocyte precursor cells via plastic adherence. Isolated SVF cells were cultivated in DMEM/Ham F-12 culture medium containing 10% FBS and 100 U penicillin and 0.1 mg ml−1 streptomycin (Sigma-Aldrich) at 37 °C and 5% CO2 and passaged every 3–4 d.
6
Whole Blood Immune Cell Phenotyping
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Samples were processed as follows: 1,6ml of whole blood was collected in Monovette EDTA tubes (Sarstedt, Germany) and analyzed 24 hours post collection latest. 100l blood of each patient was collected in a 96-well plate (Greiner Bio-one, Germany) and diluted 1:1 with phosphate buffered saline (Dulbecco’s PBS, Sigma Aldrich, United Kingdom). After centrifugation for 4 minutes at 4°C and 400g samples were lysed twice with 180μl ACK buffer (aqueous solution of 150mM NH4Cl (Sigma Aldrich, Germany), 10mM KHCO3 (Merck KGaA, Germany) and 0.1mM EDTA (Merck KGaA, Germany) adjusted to pH = 7,3) for ten minutes. Cells were washed with FACS buffer (phosphate buffered saline (Dulbecco’s PBS, Sigma Aldrich, Germany), 2% fetal bovine serum (Gibco, life technologies, Germany), 0.01% NaN3 (Serva GmbH, Germany and Karl Roth GmbH, Germany) and 2mM EDTA (Merck KGaA, Germany) and incubated with the monoclonal antibody mix for 30 minutes at 4°C in the dark. The samples were washed again and resuspended in FACS buffer for analysis. Flow cytometry was performed on the CyAn ADP (Dako, Agilent Technologies). Samples from the Technische Universität München were processed as described above with minor differences in the composition of buffers and a shorter incubation time of monoclonal antibodies [13 (link)].
7
Quantification of Acetylcholine and Choline
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Acetylcholine (ACh) and choline (Ch) in dialysates were determined by microbore HPLC-ECD, using the Eicom HTEC-500 system (Kyoto, Japan) equipped with low-speed pump, pre- and separation column, enzyme reactor carrying immobilised AChE and choline oxidase, and electrochemical detector with a platinum electrode operating at 500 mV. The mobile phase consisted of KHCO3 50 mmol/L (Merck, Darmstadt, Germany), EDTA-2Na 134.3 μmol/L (BDH, Poole, UK) and sodium decane-1-sulfonate 1.64 mmol/L (Alfa Aesar, Karlsruhe, Germany) in RotisolV HPLC gradient grade water (Sigma Aldrich, Munich, Germany), brought to pH 8.4. The flow rate was 150 μL/min. At an injection volume of 5 μL, the detection limit of this system was 1–2 fmol/injection. Intra-assay and inter-assay coefficients of variability have previously been described [16 (link)]. Data acquisition was performed using the EPC-500 PowerChrom® software.
Acetylcholinesterase activity was measured in brain homogenates using the Ellman assay with minor modifications as described [17 (link)].
Acetylcholinesterase activity was measured in brain homogenates using the Ellman assay with minor modifications as described [17 (link)].
8
Immunophenotyping of Tumor Cells
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Blood samples were taken routinely from the retrobulbar venous plexus of vaccinated and control mice. Blood samples were stained with a panel of conjugated monoclonal antibodies (mAb, 1 μg each) followed by lysis of erythrocytes (155 mM NH4Cl (MERCK Millipore, Darmstadt, Germany), 10 mM KHCO3 (MERCK Millipore), 0.1 mM EDTA (Applichem, Darmstadt, Germany). Intracellular staining was done upon incubation with 1x Intracellular Staining Perm Wash Buffer (Biolegend, Koblenz, Germany). Negative controls consisted of lymphocytes stained with the appropriate isotypes (Biolegend). Cultured tumor cell lines were phenotyped with FITC-, PE-, APC-, PE-Cy7-, or APC-Cy7-labeled mAbs as follows: PD1, PD-L1, CTLA-4, LAG-3, TIM-3, IDO-1, IFN-γ, and TNF-α (Biolegend). Cells were washed, resuspended in PBS and analyzed by flow cytometry on a FACS Verse Cytometer (BD Pharmingen). Data analyses were performed using BD FACSuite software (BD Pharmingen).
9
Multicolor Flow Cytometric Analysis
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Blood samples were taken routinely from the retrobulbar venous plexus. Single cell suspensions of spleens and GIT were obtained upon passing them through a cell strainer (100 µm). Samples (2 × 105/Well) were stained with a panel of conjugated monoclonal antibodies (mAb, 1 μg each) followed by lysis of erythrocytes (155 mM NH4Cl (MERCK Millipore, Darmstadt, Germany), 10 mM KHCO3 (MERCK Millipore) and 0.1 mM EDTA (Applichem, Darmstadt, Germany). Negative controls consisted of lymphocytes stained with the appropriate isotypes (Biolegend, San Diego, USA). Cells were washed, resuspended in PBS and analyzed by flow cytometry on a Flow Cytometer (BD FACSVerse™, BD Pharmingen). Data analysis was performed using BD FACSuite software (BD Pharmingen).
10
Immunophenotyping of Tumor Cells
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Surface marker expression on single tumor cell suspensions was assessed by multi-color flow using a panel of human-specific conjugated antibodies (mAb, 1 μg each): anti-CD3 FITC (clone OKT-3), anti-CD4 PE (clone IT4), anti-CD8 PE (clone MEM-31), anti-CD56 PE (clone MEM-188), anti-CD16 APC (clone 3G8), anti-CD274 PECy7 (clone 29E.2A3), anti-CD70 FITC (clone 113–16), anti-CD14 FITC (clone 63D3), anti-CD204 PE (clone 7C9C20), anti-CD169 APC (clone 7–239), anti-CD163 PECy7 (clone GHI/61). Whole blood and tumor samples were stained for 30 min (4 °C). Afterwards, erythrocytes were lysed using 155 mM NH4Cl (MERCK Millipore, Darmstadt, Germany), 10 mM KHCO3 (MERCK Millipore), and 0.1 mM EDTA (Applichem, Darmstadt, Germany). Negative controls were stained with the appropriate isotypes (Biolegend) or left unstained. Cells were washed, resuspended in PBS and analyzed by flow cytometry on a FACSVerse Cytometer (BD Pharmingen). Data analysis was performed using BD FACSuite software (BD Pharmingen).
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