PBMCs obtained from three healthy adult subjects were purchased from AllCells (Alameda, CA, USA). PBMCs were seeded at a density of 5 × 104 cells per well in a 96-well plate. The culture medium was RPMI 1640 with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich). The cells were cultured at 37 °C with 5% CO2 for 24 h in media containing the following adjuvant concentrations: 10 μg/ml WPV, 100 μg/ml alum, 20 μg/ml R848, 20 μg/ml poly I:C, 5% AddaVax, 0.03% or 0.3% CVP, and 50 μg/ml CpG ODN 2006. Post incubation, culture supernatants were centrifuged at 3000 × g for 10 min at 4 °C, and the supernatants were collected for cytokine analysis. The concentration of cytokines and chemokines in supernatants were measured using the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel Immunology Multiplex Assay (Merck Millipore).
Penicillin
Manufactured by Merck Group
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About the product
Penicillin is a type of antibacterial drug that is widely used in medical and laboratory settings. It is a naturally occurring substance produced by certain fungi, and it is effective against a variety of bacterial infections. Penicillin works by inhibiting the growth and reproduction of bacteria, making it a valuable tool for researchers and medical professionals.
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16 427 protocols using penicillin
1
Evaluating Cytokine Responses to Adjuvants
2
Osteogenic Differentiation of MC3T3-E1 Cells
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MC3T3-E1 cells were maintained in high glucose medium (HyClone, Logan, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; BBI, Shanghai, China), 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone, Logan, USA) and cultured with 5% CO2 at 37 °C. For osteogenic differentiation, the cells were cultured in high glucose medium supplemented with 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10− 8 mol/L dexamethasone (Sigma, St, Louis, MO) and 10 mmol/L β-glycerophosphate (Solarbio, Beijing, China). The medium was replaced every two days.
Referring to past studies, ephrinB2-fc (Sino Biological, Beijing, China) was used to stimulate ephrinB2-Ephb4 signaling in MC3T3-E1 cells at a concentration of 4 µg/ml(Wang et al. 2018). NVP-BHG712 (Selleck, Shanghai, China) was used to inhibit Ephb4 signaling, and we used it at a concentration of 200 nM(Mundy and Elefteriou 2006).
Referring to past studies, ephrinB2-fc (Sino Biological, Beijing, China) was used to stimulate ephrinB2-Ephb4 signaling in MC3T3-E1 cells at a concentration of 4 µg/ml(Wang et al. 2018). NVP-BHG712 (Selleck, Shanghai, China) was used to inhibit Ephb4 signaling, and we used it at a concentration of 200 nM(Mundy and Elefteriou 2006).
3
Cytotoxicity Assessment of Hydrogels
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The cytotoxicity of different hydrogels was assessed using a CCK-8 assay against L929 cells. In brief, the MACS, BC, and BC-MACS hydrogels were cultured in the DMEM medium supplemented with 10% (v/v) FBS (Gibco, Invitrogen, Carlsbad, CA, USA), 100 IU mL−1 of penicillin, and 100 μg mL−1 of streptomycin (Sigma-Aldrich, Gillingham, UK) at 37 °C for 24 h. The DMEM medium of the overnight cultured L929 cells was replaced with 100 μL of hydrogel extract medium. After 24 h of incubation, CCK-8 solution (10 μL) was added to each well and continuously incubated for 4 h. The absorbance at 450 nm was recorded using a microplate reader. The percentage of viability was calculated as a percentage of the untreated control cells.
4
Cell Culture Protocols for Various Cell Lines
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The mouse leukemic monocyte macrophage cell line (RAW 264.7—ATCC number TIB-71) was cultured in DMEM medium (pH 7.2) supplemented with fetal bovine serum (FBS, 10%) (both from Life technologies, Carlsbad, CA, USA), glucose (up to 25 mM), sodium bicarbonate (17.95 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) (all from Sigma-Aldrich, St. Louis, MO, USA).
Mouse skin fibroblasts (3T3, obtained from ATCC CRL-1658) and the human keratinocyte cell line (HaCaT, CLS 300493, acquired from DKFZ) were grown in DMEM medium (pH 7.2) supplemented with heat-inactivated FBS (10%) (both from Life technologies, Carlsbad, CA, USA), glucose (up to 25 mM), sodium bicarbonate (35.9 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) (all from Sigma-Aldrich, St. Louis, MO, USA).
Mouse melanocytes established from a B16 melanoma tumor (B16V cells; DSMZ ACC-370, Braunschweig, Germany) were cultured in DMEM medium (pH 7.2) containing glucose (5.6 mM) and sodium bicarbonate (44 mM), supplemented with heat-inactivated FBS (10%), 50 U/mL penicillin, and 50 μg/mL streptomycin (all from PAN-Biotech GmbH, Aidenbach, Germany).
The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Before reaching confluence, the cells were detached with a cell scraper (RAW 264.7), or with trypsin (3T3, HaCaT and B-16V) and further subcultured in fresh culture media.
Mouse skin fibroblasts (3T3, obtained from ATCC CRL-1658) and the human keratinocyte cell line (HaCaT, CLS 300493, acquired from DKFZ) were grown in DMEM medium (pH 7.2) supplemented with heat-inactivated FBS (10%) (both from Life technologies, Carlsbad, CA, USA), glucose (up to 25 mM), sodium bicarbonate (35.9 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) (all from Sigma-Aldrich, St. Louis, MO, USA).
Mouse melanocytes established from a B16 melanoma tumor (B16V cells; DSMZ ACC-370, Braunschweig, Germany) were cultured in DMEM medium (pH 7.2) containing glucose (5.6 mM) and sodium bicarbonate (44 mM), supplemented with heat-inactivated FBS (10%), 50 U/mL penicillin, and 50 μg/mL streptomycin (all from PAN-Biotech GmbH, Aidenbach, Germany).
The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Before reaching confluence, the cells were detached with a cell scraper (RAW 264.7), or with trypsin (3T3, HaCaT and B-16V) and further subcultured in fresh culture media.
5
Culturing MCF-7 Breast Cancer Cells
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The MCF-7 breast cancer cell lines used in this study were obtained from NCCS, Pune, India. These cell lines were maintained in DMEM supplemented with L-glutamine, 10% (v/v) FBS and 1% (v/v) antibiotic solution (100 U/mL penicillin and 1000 μg/mL streptomycin, Sigma), as previously described. The cells were cultured at 37 °C in a humidified environment with 95% air and 5% carbon dioxide (CO2). When the cells reached confluence, the first step in preparing them for further processing involved carefully removing the culture medium from the flask (corning). To ensure the removal of any residual medium or contaminants, the cells were then rinsed twice with sterile PBS. The confluent layer of cells was subsequently trypsinized using Trypsin/EDTA (Gibco) and resuspended in a fresh medium (DMEM) for subculture83 (link).
6
Cytotoxicity and Apoptosis Assays
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The 2,2-diphenyl-1-picrylhydrazyl (DPPH),ethidium bromide and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Dulbecco’s modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI-1640), minimal essential media (MEM), fetal bovine serum (FBS), penicillin, streptomycin sulfate salt powder, glutaraldehyde, acridine orange, propidium iodide, 2,7-dichloro dihydro fluorescein diacetate (DCFH-DA), rhodamine 123 (Rh123), Hoechst 33342 and fluoromount were obtained from Sigma-Aldrich (St. Louis, MO, USA). The remaining chemicals and reagents used for experimental analysis were of analytical grade.
7
Cell Line Cultivation and Authentication
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K1 cells were obtained from Sigma Aldrich. The ATC cell line, THJ-16T, was obtained from Dr John Copland (Mayo Clinic). WPMY-1 and HEK293 (CRL-1573) cells were obtained from American Type Culture Collection (ATCC). Cells were authenticated using STRS analysis and maintained and used experimentally at fewer than 20 passages from thaw. K1 cells were grown in RPMI (VWR) containing 10% fetal bovine serum (ThermoFisher Scientific), 1% penicillin-streptomycin (100 units/mL penicillin and 0.1 mg/mL streptomycin, Sigma), 1X MEM Non-Essential Amino Acids (VWR), and 1 mM sodium pyruvate (Vanderbilt Molecular Biology Resource). WPMY −1 and HEK293 cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (Sigma) containing 8% to 10% fetal bovine serum (ThermoFisher Scientific), and 1% penicillin-streptomycin (100 units/mL penicillin and 0.1 mg/mL streptomycin, Sigma). All cell lines tested negative for mycoplasma contamination.
8
HEK-293FT Cell Culture and siRNA Knockdown
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HEK-293FT cells (ThermoFisher Scientific) were cultured in DMEM (Corning Inc.) supplemented with 10 % FBS, 2mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Sigma-Aldrich) and maintained at 37 °C, 5 % CO2 in a humidified incubator. For RNA interference, cells were transfected with Lipofectamine RNAiMAX (ThermoFisher Scientific) following the manufacturer’s specifications. Transfected siRNA sequences were as follows: siRNA CEBPZ_1: rCrArArArArGrUrCrArGrUrArCrUrArArArArArArArGrCAA; siRNA CEBPZ_2: rUrUrGrCrUrUrUrUrUrUrArGrUrArCrUrGrArCrUrUrGrArG; negative control: scrambled negative control DsiRNA, all from Integrated DNA Technologies). 72 hours after transfection, the cells were harvested, and total RNA was extracted.
9
Neuroblastoma Cell Culture and Treatments
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Human neuroblastoma SKNAS cells were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). The NXS2 murine neuroblastoma cell line was kindly provided by Holger N. Lode (University Medicine Greifswald, Germany) [20 (link)]. SKNAS, parental NXS2 cells, and stable-transduced NXS2 CTRL and hLGALS3BP cells were all cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco; Whaltman, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich Corporation, St. Louis, MO, USA). All cells were maintained at 37 °C in a humidified air with 5% CO2. All cell lines were tested for mycoplasma contamination through polymerase chain reaction (PCR).
SH-DM4 was purchased from MedChemExpress (HY-12454; New Jersey, USA). Oxaliplatin was purchased from Sigma-Aldrich (#O9512). Anti-mouse PD-1 monoclonal antibody (InVivoMAb, CD279; clone RMP1-14, #BE0146) was purchased from BioXCell (NH, USA).
SH-DM4 was purchased from MedChemExpress (HY-12454; New Jersey, USA). Oxaliplatin was purchased from Sigma-Aldrich (#O9512). Anti-mouse PD-1 monoclonal antibody (InVivoMAb, CD279; clone RMP1-14, #BE0146) was purchased from BioXCell (NH, USA).
10
Cell Culture and Virus Packaging Protocols
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HEK293T cells (laboratory stock), Platinum-E cells (Cell Biolabs), and 293FT cells (Thermo Scientific) were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (Sigma–Aldrich) supplemented with fetal calf serum (10%) (Sigma–Aldrich), streptomycin (0.1 mg/ml) (Sigma–Aldrich), penicillin (100 U/ml), sodium pyruvate (1 mM) (Sigma–Aldrich), HEPES (10 mM) (Sigma–Aldrich), and l -glutamine (2 mM) (Sigma–Aldrich). Cells were routinely tested for mycoplasma contamination. Transfections of HEK293T and 293FT cells were performed using X-tremeGENE 9 DNA Transfection Reagent (Roche). Murine stem cell viruses (MSCVs) were packaged using Platinum-E cells by co-transfection of MSCV retroviral transfer plasmids with pCL-Eco (Addgene #12371) using X-tremeGENE 9 DNA Transfection Reagent (Roche). Lentiviruses were packaged using 293FT cells by co-transfection of lentiviral transfer plasmids with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) using X-tremeGENE 9 DNA Transfection Reagent (Roche).
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