Cells were plated at 1.4x105 cells/well in a 12-well plate and transduced the next day with FcγRIa or Fluc-encoding lentiviruses as described above. Two days post-transduction, cells were infected with wild type Lm for 1 hr (MOI = 0.015, 0.05, 0.1), washed with medium, supplemented with 50 μg/ml gentamicin (Quality Biological), and then gently overlaid with 1.5ml/well of DMEM, containing with 10% FBS, 0.4% agarose, and 20 μg/ml gentamicin (Quality Biological). The overlay was allowed to stabilize for 15 min at room temperature, when plates were moved back to an incubator at 37°C. Foci of Lm infection were visualized 30 h after initial infection by adding 200μl of 5mg/ml (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (tetrazolium MTT) (Sigma) solution to each well and incubating at 37°C for 3 h. Plates were scanned and foci of infection quantified using ImageJ software.
Gentamicin
Manufactured by Quality Biological
Gentamicin is a broad-spectrum aminoglycoside antibiotic used in laboratory settings. It functions as an inhibitor of bacterial protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Quality Biological (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Cyclosporin a, by Merck Group (2 mentions)
Gmem media, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Ciprofloxacin, by US Biological (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Tetrazolium mtt, by Merck Group (1 mentions)
Ht1080, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Rpmi 1640 media, by Corning (1 mentions)
Ciprofloxacin hydrochloride, by US Biological (1 mentions)
Hank s buffered salt solution, by Corning (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Trypsin, by Corning (1 mentions)
Human epidermal growth factor (hegf), by Merck Group (1 mentions)
Horse serum, by Atlanta Biologicals (1 mentions)
17 protocols using gentamicin
1
Lentiviral Transduction and Listeria Infection Assay
2
Culturing Human Fibrosarcoma and Breast Carcinoma Cells
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Human fibrosarcoma HT1080 cells (obtained from ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Mediatech) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone Laboratories), and 0.005% (w/v) gentamicin (Quality Biological). Human breast carcinoma MDA-MB-231 cells (obtained from ATCC) were cultured in DMEM (Mediatech) supplemented with 10% FBS (Hyclone) and 1% penicillin-streptomycin. The cells were maintained at 37° C and 5% CO2 in a humidified incubator during cell culture and live-cell microscopy. All cell lines were tested for mycoplasma and deemed free of contamination.
3
Cell Culture of Prostate Cancer Lines
4
Cell Culture Conditions for Various Cell Lines
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Human fibrosarcoma HT1080 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Mediatech) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone Laboratories), and 0.005% (w/v) gentamicin (Quality Biological). Human breast carcinoma MDA-MB-231 cells (ATCC) and MCF-7 cells (ATCC) were cultured in DMEM (Mediatech) supplemented with 10% FBS (Hyclone). Human glioblastoma U-87 MG cells (ATCC) were cultured in DMEM (Mediatech) supplemented with 10% FBS (Hyclone). Human diploid cell line, WI-38, (ATCC) were cultured in Eagle's minimal essential medium (EMEM, Mediatech) supplemented with 10% FBS (Hyclone). Human breast epithelial MCF10A cells (ATCC) and MCF12A cells (ATCC) were cultured in DMEM supplemented with 5% horse serum (Atlanta biologicals), 20 ng ml−1 Human epidermal growth factor (Sigma-Aldrich), 100 ng ml−1 cholera toxin, (Sigma-Aldrich) 0.01 mg ml−1 bovine insulin (Life technologies), and 500 ng ml−1 hydrocortisone (Sigma-Aldrich). HT1080 cells transfected with shRNAs (see below) were grown in medium containing 1 μg ml−1 puromycin. The cells were maintained at 37 °C and 5% CO2 in a humidified incubator during cell culture and during live-cell microscopy. All cell lines were tested for mycoplasma and deemed free of contamination.
5
Generating Lymphoblastoid Cell Lines
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SLCLs were generated by spontaneous expansion of PBMCs from HC donors and patients with MS in the presence of cyclosporin A.32 (link) In brief, PBMCs were thawed in growth medium comprising RPMI 1640 (Gibco, Gaithersburg, MD) with 10% fetal bovine serum (FBS; Gibco), 1% gentamicin (50 mg/mL; Quality Biological, Gaithersburg, MD), and 1% l -Glutamine (200 mM; Quality Biological). PBMCs were rested overnight and then were plated into 96-well plates (round bottom) at a density of 1 ×x 106/well in a 200-µL total volume. A minimum of 10 wells to a maximum of 30 wells were prepared for each sample according to the available PBMCs of controls and patients. Once a week, 100 µL of medium was removed and replaced with fresh growth medium. After 21 days, cyclosporin A (2 µg/mL; Sigma-Aldrich, Inc., St. Louis, MO) was added to the wells. Three to 6 weeks later, clusters of B-lymphoblastoid cells started to develop. Those clusters were then collected in bigger wells until further expanded in T25 flasks and maintained in growth medium at 37°C under a 5% CO2-humidified atmosphere. PCR for a known deletion in EBV laboratory strain B95.8 was used to confirm that the lines were transformed with endogenous EBV, and ddPCR was used to confirm the EBV infection in the newly generated SLCLs.
6
Cell culture protocols for Vero and BSR T7/5 cells
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African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies). Vero cells were maintained in 2% FBS during experiments. Baby hamster kidney cells that constitutively express the T7 polymerase (BSR T7/5) [43 (link)] were grown in GMEM media (Gibco-Life Technologies) with 10% FBS and 2% non-essential amino acids (Gibco-Life Technologies). Every other cell passage, 2% of gentamicin (Quality Biological) was added to the media to maintain selection for the T7-polymerase-expressing cells. Both cell lines were maintained at 37°C with 5% CO2.
7
Vero and BSR T7/5 Cell Culture
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African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) supplemented with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies) at 37°C with 5% CO2. Baby hamster kidney cells constitutively expressing the T7 polymerase (BSR T7/5) [56 (link)] were grown in GMEM media (Gibco-Life Technologies) supplemented with 10% FBS and 2% MEM amino acids (Gibco-Life Technologies). Every second cell passage, 2% of gentamicin (Quality Biological) was added to maintain selection for the T7-polymerase-expressing cells.
8
Thawing and Culturing Frozen TILs
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Frozen TILs were thawed in complete media containing RPMI1640 medium (Lonza), 10% heat-inactivated human AB serum (catalog no. HP1022I, Valley Biomedical Inc.), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies), 2 mmol/L l -glutamine (Life Technologies), 10 μg/mL gentamicin (Quality Biological Inc.), 12.5 mmol/L HEPES (Life Technologies) with 6,000 IU recombinant human IL2 (Proleukin, Prometheus Laboratories Inc.) at 37°C in 5% CO2. All other healthy donor lymphocytes were cultured in the same media with 300 IU IL2.
9
Cytotoxicity Assay for UGT Inhibitors
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We measured cytotoxicity of the UGT inhibitors using a Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific). HEK cells were seeded at 5 x 104 per well in DMEM (Quality Biological) with 10% Hyclone Cosmic Calf Serum (Thermo Fischer), 200 μM of L-glutamine (Quality Biological), and 50 μg/mL of gentamicin (Quality Biological) at 37°C in 5% CO2. We then incubated the cells with various concentrations of the UGT inhibitors overnight. Following this, we transferred 50 μL of media from each well to a new 96-well plate and then added 50 μL of reaction buffer. We incubated the mixture for 30 minutes and then added 50 μL of stop solution. We measured absorbance at 490 nm and 680 nm. We employed the following controls: a spontaneous LDH activity control which was incubated with the vehicle only and a maximum LDH activity control which was incubated with nothing but later lysed prior to incubation with the reaction buffer. We calculated absorbance for each well by subtracting the 680 nm absorbance value (background) from the 490 nm absorbance value. We then calculated percent cytotoxicity using the following equation:
10
Splenic Immune Cell Stimulation Assay
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A single-cell suspension was prepared from spleens by mechanical homogenization and straining through a 22μm cell-strainer. RBCs were lysed using ACK lysis buffer (Quality Biological) and 2.0x106 were plated in IMDM media (Cellgro) containing 1% L-glutamine, 10% FCS, 1% insulin-transferrin-selenium and 80μg/mL gentamicin (Quality Biological). Cells were stimulated with either media, 5μg/mL anti-CD3 and 2μg/mL anti-CD28, or 20μg/mL LsAg and cultured at 37°C, 5% CO2 for 3 days. Studies in which purified CD4+ and CD11C+ were used, cells were magnetically isolated by positive selection according to manufacturer’s instructions (Miltenyi Biotech). Purified CD4+ cells were cultured with purified CD11c+ cells in a 1:10 ratio (CD11c+: CD4+). Cells were stimulated as outlined above. ELISAs were performed on culture supernatants. IL-4, IL-5, IFN-γ and IL-10 were detected in culture supernatants using Costar half-area plates and BD OptEIA ELISA kits (BD Biosciences) following the manufacturer’s instructions.
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