The A549 human non‐small cell lung cancer cell line, the human HL‐60 promyelocytic leukemia cell line, and the 4T1 murine triple negative breast cancer cell line was purchased from the American Type Culture Collection. Cells were maintained in the following media: A549 cells in Dulbecco's modified Eagle medium (DMEM)/F12 (containing 3.151 g/L glucose), RPMI (containing 2 g/L glucose) for the HL‐60 or 4T1 cell lines. Media were purchased from Capricorn Scientific GmbH and supplemented with 10% fetal bovine serum (Euroclone, Peri), 2 mM GlutaMAX (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin antibiotics (penicillin G sodium salt and streptomycin sulfate salt; Merck). The cells were cultured in a standard tissue culture Petri dish, 10 mm in diameter (Corning Life Sciences) at maximum 80% confluence in a standard atmosphere of 95% air and 5% CO2 (Sanyo).
Streptomycin antibiotic
Manufactured by Merck Group
Sourced in United States, Germany
About the product
Streptomycin is a broad-spectrum antibiotic produced by the bacterium Streptomyces griseus. It is used to treat a variety of bacterial infections. Streptomycin works by inhibiting protein synthesis in bacterial cells, leading to their death or inhibition of growth.
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Lab products found in correlation
Penicillin, by Merck Group (11 mentions)
Dmem medium, by Merck Group (3 mentions)
Infinite m200, by Tecan (2 mentions)
F7524, by Merck Group (1 mentions)
L glutamine solution, by Merck Group (1 mentions)
Sodium chloride (nacl), by Balkanpharma-Troyan (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Hemocytometer, by Boeco (1 mentions)
Mcf 7, by Biologix (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Infinite m200 spectrophotometer, by Tecan (1 mentions)
Penicillin streptomycin antibiotic, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Dmem medium, by Euroclone (1 mentions)
Red cmtpx fluorescent dye, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Cellsense entry software, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Dutscher (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Anti gfp vhh g4, by Addgene (1 mentions)
Check compatibility
14 protocols using streptomycin antibiotic
1
Cell Line Culture Protocol for Cancer Research
2
Inducible Expression of Fluorescent Septin Fusions
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U2OS osteosarcoma cells for the expression of full-length fluorescent protein septin fusions were from ATCC (HBT-96). For the inducible co-expression of β10- and β11-tagged septins in the context of the tripartite split-GFP complementation system, we generated an inducible U2OS-Tet-On-GFP1-9 cell line which expresses constitutively a GFP1-9 fragment and an anti-GFP VHH intrabody that enhances split-GFP fluorescence. To generate this cell line, U2OS cells were successively transduced with lentiviruses encoding rtTA, GFP1-9 (#130271; Addgene) and anti-GFP VHH G4 (#182236; Addgene) and tested for complementation efficiency using transient expression of a GFP10-zipper-GFP11 domain (Koraïchi et al., 2018 (link)). One additional round of transduction with GFP1-9 lentivirus led to an optimized U2OS-Tet-On-GFP1-9 cell line that showed 80% GFP positive cells upon expression of the GFP10-zipper-GFP11 domain. An IRES-TagBFP cassette downstream β10-tagged septins was used for monitoring septin expression. Cells were maintained in McCoy’s medium (16600082; Gibco) supplemented with 10% fetal bovine serum (S181H; Dominique Dutscher), 100 U/ml penicillin and 100 μg/ml streptomycin antibiotics (P4333; Sigma-Aldrich) in a humidified atmosphere at 37°C containing 5% CO2.
Transfections with pcDNAs, for the screening of β10- and β11-tag combinations (Fig. S2, A–C ), were performed 16 h prior to immunostainings using jetPRIME (101000015; PolyPlus). To obtain single cells for imaging, 50 × 103 U2OS-Tet-On-GFP1-9 cells were typically grown on 18 mm coverslips (Knittel Glass MS0010), previously cleaned by sonication in 70% ethanol, and placed into a 12-well plate a day prior to the day of transfection, for allowing an optimal number of cells to attach and spread. A total of 0.4 μg of DNA and a 4:1 ratio of jetPRIME (μl) : DNA (μg) were used per reaction. To minimize septin overexpression artifacts, the total amount of DNA was composed by 30 ng of β10-septin, 30 ng of β11-septin and 340 ng of empty vector.
Transfections with either pCMV or pTRIP TRE Bi plasmids and siRNAs were performed through electroporation using the Neon Transfection System (MPK5000; Thermo Fisher Scientific). For pCMVs, a single 100-μl reaction using 1.8 × 106 U2OS cells, 300 pmol of each siRNA and 6 μg of each DNA was electroporated within the dedicated tip (MPK10096; Thermo Fisher Scientific). Electroporation parameters consisted in four pulses of 10 ms width and a voltage of 1,230 V. The electroporated cells were then inoculated in 5 ml of culture medium without antibiotics, and immediately divided for native-PAGE, SDS-PAGE/Western blots and immunostaining as follows: 3 ml in a 6-cm dish containing 2 ml of medium without antibiotics, 2 ml in a 6-cm dish containing 3 ml of medium without antibiotics, and 100 μl in the well of a 12-well plate containing a 18-mm coverslip in 900 μl of medium without antibiotics, respectively. A satisfactory septin knockdown efficiency was achieved within 48–96 h after electroporation. Typically, immunostaining and protein extraction were performed 72 h post electroporation.
For pTRIP TRE Bi plasmids, a single 100-μl reaction using 1.8 × 106 U2OS-Tet-On-GFP1-9 cells, 300 pmol of each siRNA and 6 μg of each DNA was electroporated within the dedicated tip using the same electroporation parameters described previously. The electroporated cells were then inoculated in 5.5 ml of culture medium without antibiotics and immediately divided for SDS-PAGE/Western blots, immunostaining and live cell imaging as follows: 5 ml in a 6-cm dish, 400 μl in the well of a 12-well plate containing a 18-mm coverslip in 600 μl of medium without antibiotics, and 200 μl in the well of a 24-well glass bottom plate (Cellvis, P24-1.5H-N) containing 800 μl of medium without antibiotics, respectively. After either 48 h, for samples intended for live cell imaging or immunostainings, or 72 h, for samples for biochemical analysis, protein expression was induced using 1 μg/ml of doxycycline (D9891; Sigma-Aldrich) for 16 h.
Transfections with pcDNAs, for the screening of β10- and β11-tag combinations (
Transfections with either pCMV or pTRIP TRE Bi plasmids and siRNAs were performed through electroporation using the Neon Transfection System (MPK5000; Thermo Fisher Scientific). For pCMVs, a single 100-μl reaction using 1.8 × 106 U2OS cells, 300 pmol of each siRNA and 6 μg of each DNA was electroporated within the dedicated tip (MPK10096; Thermo Fisher Scientific). Electroporation parameters consisted in four pulses of 10 ms width and a voltage of 1,230 V. The electroporated cells were then inoculated in 5 ml of culture medium without antibiotics, and immediately divided for native-PAGE, SDS-PAGE/Western blots and immunostaining as follows: 3 ml in a 6-cm dish containing 2 ml of medium without antibiotics, 2 ml in a 6-cm dish containing 3 ml of medium without antibiotics, and 100 μl in the well of a 12-well plate containing a 18-mm coverslip in 900 μl of medium without antibiotics, respectively. A satisfactory septin knockdown efficiency was achieved within 48–96 h after electroporation. Typically, immunostaining and protein extraction were performed 72 h post electroporation.
For pTRIP TRE Bi plasmids, a single 100-μl reaction using 1.8 × 106 U2OS-Tet-On-GFP1-9 cells, 300 pmol of each siRNA and 6 μg of each DNA was electroporated within the dedicated tip using the same electroporation parameters described previously. The electroporated cells were then inoculated in 5.5 ml of culture medium without antibiotics and immediately divided for SDS-PAGE/Western blots, immunostaining and live cell imaging as follows: 5 ml in a 6-cm dish, 400 μl in the well of a 12-well plate containing a 18-mm coverslip in 600 μl of medium without antibiotics, and 200 μl in the well of a 24-well glass bottom plate (Cellvis, P24-1.5H-N) containing 800 μl of medium without antibiotics, respectively. After either 48 h, for samples intended for live cell imaging or immunostainings, or 72 h, for samples for biochemical analysis, protein expression was induced using 1 μg/ml of doxycycline (D9891; Sigma-Aldrich) for 16 h.
3
MTT Assay for Cell Viability Analysis
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The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was carried out to determine the influence of the MBGNs on the cell proliferation, and viability. In viable cells, nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes are directly involved in the mitochondrial metabolization process of the MTT. Hence, as a result of the enzymes conversion, the water-soluble yellow MTT is transformed into purple insoluble formazan. The obtained salt is then dissolved in isopropanol and the optical density of the solution is spectrophotometrically quantified. A higher value of the collected signal corresponds to an increase of the cell proliferation, signal which is directly proportional to the cell viability.
In this study Vybrant™ MTT Cell Viability Assay (Thermo Fisher Scientific, Waltham, MA, USA) together with the THP-1 human leukaemia monocytic cells were used for the MTT assay. Firstly, the sterilised MBGNs disks were place separately in each well of 96-well plates, followed by cell seeding at a density of 3000 cells/well. DMEM medium (Sigma-Aldrich, Missouri, MO, USA), 10% fetal bovine serum and 1% streptomycin antibiotics (Sigma–Aldrich, Missouri, MO, USA) were the components of the growing medium. For the control group, the THP-1 cells were grown in DMEM, 10% fetal bovine and 1% streptomycin/neomycin, penicillin. Afterwards, they were treated the same way as the groups exposed to MBGNs. While the cells were incubated at 37 °C in the presence or not of the MBGNs for 24 h and 72 h, the culture medium was changed twice a week to assure that the optimal growing conditions were fulfilled. Once the established testing time passed, 15 mL of MTT was added in each well. After another 4 h of incubation, 10 mL of a solution containing 1 mg of sodium dodecyl sulphate and 10 ml HCl (0.01 M) was subsequently added. When the formazan crystals became solubilised, TECAN Infinite M200 spectrophotometer (Männedorf, Switzerland) was used to quantify the optical density of the obtained solution at 570 nm. These results were further used together with Equation (1) to determine the relative cell viability.
In this study Vybrant™ MTT Cell Viability Assay (Thermo Fisher Scientific, Waltham, MA, USA) together with the THP-1 human leukaemia monocytic cells were used for the MTT assay. Firstly, the sterilised MBGNs disks were place separately in each well of 96-well plates, followed by cell seeding at a density of 3000 cells/well. DMEM medium (Sigma-Aldrich, Missouri, MO, USA), 10% fetal bovine serum and 1% streptomycin antibiotics (Sigma–Aldrich, Missouri, MO, USA) were the components of the growing medium. For the control group, the THP-1 cells were grown in DMEM, 10% fetal bovine and 1% streptomycin/neomycin, penicillin. Afterwards, they were treated the same way as the groups exposed to MBGNs. While the cells were incubated at 37 °C in the presence or not of the MBGNs for 24 h and 72 h, the culture medium was changed twice a week to assure that the optimal growing conditions were fulfilled. Once the established testing time passed, 15 mL of MTT was added in each well. After another 4 h of incubation, 10 mL of a solution containing 1 mg of sodium dodecyl sulphate and 10 ml HCl (0.01 M) was subsequently added. When the formazan crystals became solubilised, TECAN Infinite M200 spectrophotometer (Männedorf, Switzerland) was used to quantify the optical density of the obtained solution at 570 nm. These results were further used together with Equation (1) to determine the relative cell viability.
4
Culturing MCF-7 Breast Cancer Cells
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Human breast cancer cells (MCF-7) were purchased from Cell Bank of Iran, Pasteur Institute of Iran, Tehran, I.R. Iran. MCF-7 cells were seeded in a flask (Biologix-TC75) containing 12 mL of the medium of the completed Dulbecco’s modified eagle (DMEM- F12, Gibco, USA) with 10% fetal bovine serum (FBS), 10 μg/mL penicillin, and streptomycin antibiotics (Sigma, USA) at 37 °C, 5% CO2 conditions. The medium was changed three times per week. The cells were subcultured regularly after 80-90% confluency using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Cat No: N0100-780, Cegrogen, Germany) and counted by a hemocytometer (Boeco, Germany). ADSCs, isolated from adipose tissue in the previous study (Ethics No. IR.MUI.MED.REC.1399.1171), were thawed and cultured (21 (link)).
5
Cell Culture and Electroporation Protocols
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The cell culture medium consisted of Dulbecco‘s modified Eagle‘s medium (cat. No. D5546, Sigma-Aldrich Chemie, Steinheim, Germany), 9% fetal bovine serum—FBS (F7524, Sigma-Aldrich Chemie), 1% L–glutamine solution (G7513, Sigma-Aldrich Chemie), 100 U/mL penicillin, and 90 μg/mL streptomycin antibiotics (P0781, Sigma-Aldrich Chemie). The cell electroporation medium was Minimum Essential Medium Eagle, Spinner Modification (S-MEM) (M8167, Sigma-Aldrich Chemie). Altogether, 0.9% NaCl (Balkanpharma-Troyan AD, Troyan, Bulgaria), FeCl2, and FeCl3 (Fluka Chemie GmbH, Buchs, Switzerland) were used to prepare the solutions of 0.9% NaCl with various concentrations of Fe2+ and Fe3+.
6
Culturing HT-29 and HDF 106-05 cells
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The human Caucasian colon adenocarcinoma cell line, HT-29 (Interlab Cell Line Collection, Genova, Italy), was cultured as a monolayer in RPMI-1640 growth medium (Sigma-Aldrich, Milano, Italy). The normal adult human primary dermal fibroblast cell line, HDF 106-05 (ECACC, Salisbury, UK), was cultured as a monolayer in DMEM growth medium (Sigma-Aldrich). Both cell culture media were supplemented with 10% decomplemented fetal bovine serum (FBS) (Lonza, Verviers, Belgium), 2 mM-glutamine, 100 UI/mL penicillin and 100 μg/mL streptomycin antibiotic (Sigma-Aldrich). Both cell lines were maintained in a dark incubator (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere containing 5% CO2 at 37 °C.
7
Antibacterial Screening of Strain MHSD3
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The antibacterial activity of strain MHSD3 crude extract was performed by minimum inhibition concentration (MIC) using the method described by Andrews (2001) (link) with minor modifications. The following indicator strains were used: B. cereus (ATCC 10876), Mycobacterium smegmatis (ATCC 21293), V. parvula (ATCC 10790), E. coli (ATCC 10536), P. aeruginosa (NCTC 10662), Klebsiella pneumonia (ATCC 10031), K. oxytoca (ATCC 13182), S. aureus (ATCC 25923), S. saprophyticus (ATCC 15305), S. epidermidis (ATCC 14990), and E. faecium (ATCC 13048). The indicator strains were inoculated into Mueller Hinton broth (MHB) for 24 h at 37°C. The inoculums were compared with 0.5 McFarland standard by diluting the culture with MHB. A stock solution of 0.015-g bacterial crude extract was dissolved in 1 ml of dimethyl sulfoxide to make an initial concentration of 15 mg/ml. The serial dilution from stock was carried out using MHB ranging from 15 to 0.23 mg/ml. The 96 well plates were filled with 100-µl indicator strains and 100 µl of different concentrations of crude extracts. Streptomycin antibiotic (Sigma-Aldrich) was used as a positive control and dimethyl sulfoxide as a negative control. All tests were performed in triplicates, and the plates were incubated at 37°C for 24 h. After incubation, 10 µl of 0.02% (w/v) of resazurin sodium salt solution was added to each well as an indicator of microbial growth, and plates were incubated for 2 h at 37°C. A blue color indicated growth inhibition, and a color change from blue to pink indicated bacterial growth.
8
Antimicrobial Potential of Bacterial Endophyte Extracts
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Minimum inhibitory concentration studies of the bacterial endophyte's crude extracts were performed according to the method by [16 (link)] with some modifications. Briefly, stock solutions of the extracts were prepared by dissolving 0.02 g in 1 mL dimethyl sulfoxide (DMSO) to produce a final concentration of 20 mg/mL, which were then serially diluted to concentrations of 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, and 0.312 mg/mL using Mueller-Hinton broth. The pathogenic test strains used were Gram-negative bacteria Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), and Klebsiella oxytoca (ATCC13182) and Gram-positive bacteria Staphylococcus aureus (NCTC6571) and Bacillus cereus (ATCC10876). Using McFarland 0.5 standard, 50 μL of each pathogen was inoculated in 15 mL of Muller-Hinton broth and incubated at 37°C for 24 hours. Following aseptic techniques in 96 well microtitre plates, 100 μL of pathogenic test strains was added horizontally and 100 μL of the diluted crude extract concentrations was added vertically starting from high to low concentrations. Negative controls [100 μL DMSO and 100 μL sterile Mueller-Hinton broth, 1 : 1 ratio (v/v)] and positive control 1 mg/mL Streptomycin antibiotic (Sigma-Aldrich, Switzerland) were added vertically in the wells subsequent to those with low concentrations. The microtitre plates were incubated at 37°C for 24 hours. The MIC assays were conducted in triplicates. After incubation, ten microliters of resazurin sodium salt solution [0.02% (w/v)] was added to the wells as an indicator of microbial growth and incubated for another 2 hours. The ability of the crude extracts to inhibit pathogenic strains was indicated by no colour change of resazurin sodium salt solution (remains blue) and the colour change from blue to pink after incubation indicated no bacterial inhibition. The MIC (lowest concentration of each extract displaying no visible growth) values were visually determined.
9
Melanoma and HeLa CIITA cell lines
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Melanoma cell line M121224 (52 (link)) was cultured in RPMI 1640 medium, 10% FBS, 10 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin antibiotics (Sigma, Germany).
HeLa cells stably expressing CIITA (HeLa-CIITA, kindly provided by Prof. P. Cresswell, New Haven CT, USA) were grown in DMEM medium (Euroclone), 10% FBS, 10 mM HEPES, and penicillin–streptomycin antibiotics (Sigma, Germany).
M121224 were pre-treated for 3 h with 50 ng/ml IFN-γ and both M121224 and HeLa CIITA cells were transfected with specific or scramble miRNAs encoding plasmids. After 48 h, CIITA mRNA expression was evaluated by qRT-PCR and HLA-II surface expression was evaluated by FACS analysis.
HeLa cells stably expressing CIITA (HeLa-CIITA, kindly provided by Prof. P. Cresswell, New Haven CT, USA) were grown in DMEM medium (Euroclone), 10% FBS, 10 mM HEPES, and penicillin–streptomycin antibiotics (Sigma, Germany).
M121224 were pre-treated for 3 h with 50 ng/ml IFN-γ and both M121224 and HeLa CIITA cells were transfected with specific or scramble miRNAs encoding plasmids. After 48 h, CIITA mRNA expression was evaluated by qRT-PCR and HLA-II surface expression was evaluated by FACS analysis.
10
Evaluating ZnO Nanoparticle Biocompatibility
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The human mesenchymal amniotic fluid stem cells (AFSC) were used to evaluate the biocompatibility of ZnO nanoparticles. The cells were cultured in DMEM medium (Sigma–Aldrich, Missouri, MO, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma–Aldrich, Missouri, MO, USA). To maintain optimal culture conditions, the medium was changed twice a week. The biocompatibility was assessed using MTT assay (Vybrant® MTT Cell Proliferation Assay Kit, Thermo Fischer Scientific, Massachusetts, MA, USA). The assay is a colorimetric method that allows quantitative assessment of proliferation, cell viability and cytotoxicity. The viable cells reduce yellow tetrazolium salt MTT (3- (4,5dimetiltiazoliu) -2,5-diphenyltetrazolium bromide) to a dark blue formazan via mitochondrial enzymes. Briefly, the AFSC were grown in 96-well plates, with a seeding density of 3000 cells / well in the presence of the analyzed samples (5 mg/mL concentration of each ZnO nanopowder) for 72 h. Then, 15 mL of Solution I was added and incubated at 37 °C for 4 h. Solution II was added and pipettes vigorously to solubilize formazan crystals. After 1 h, the absorbance was read using a spectrophotometer at 570 nm (TECAN Infinite M200, Männedorf, Switzerland).
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