Spleen cells obtained from mice after chemoimmunotherapy or from healthy mice, untreated MC38-bearing mice and CY-treated MC38-bearing mice were isolated and co-cultured (restimulated) for 4 days with mitomycin C-treated MC38 cells (mitomycin C were obtainted from Sigma-Aldrich) (50 mg mitomycin C/3×106 cells/ml/30 min at 37°C) in the presence of IL-2 as described (12 (link)). The source of murine IL-2 was X63mIL-2 plasmacytoma cell supernatant used at 5% concentration, corresponding to 100 U/ml. After 4 days of restimulation the cytotoxicity of the spleen cells was tested by flow cytometry. Target MC38/0 cells were stained with DiOC18 lipophilic dye (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C and then washed with PBS. After restimulation, spleen cells were incubated with labeled target cells for 4 h at 37°C at a 10:1 ratio of effector to target cells. The cells were then washed and dead cells were stained with propidium iodide (PI; Molecular Probes). Samples were analyzed using FACSCalibur flow cytometer and Flowing software 2.5.1. Percentage of cytotoxicity was determined according to formula: the percentage of dead target MC38/0 cells previously incubated with splenocytes minus the percentage of dead MC38/0 cells cultured alone.
Mitomycin c
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Macao, Japan, France, Italy, Sao Tome and Principe, China, Australia, India
Mitomycin C is a laboratory reagent used in cell biology and cancer research. It is a potent DNA cross-linking agent that inhibits DNA synthesis and cell division. Mitomycin C is commonly used to study cellular processes and as a positive control in various cell-based assays.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (73 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (62 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (42 mentions)
L glutamine, by Thermo Fisher Scientific (41 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (36 mentions)
Penicillin, by Thermo Fisher Scientific (33 mentions)
Streptomycin, by Thermo Fisher Scientific (29 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (24 mentions)
Cisplatin, by Merck Group (23 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (23 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (22 mentions)
Glutamax, by Thermo Fisher Scientific (21 mentions)
Matrigel, by BD (20 mentions)
β mercaptoethanol, by Merck Group (19 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Merck Group (17 mentions)
Etoposide, by Merck Group (17 mentions)
Trypsin edta, by Thermo Fisher Scientific (17 mentions)
Hydroxyurea, by Merck Group (16 mentions)
Dmem f12, by Thermo Fisher Scientific (15 mentions)
1 351 protocols using mitomycin c
1
Cytotoxicity Assay for Chemoimmunotherapy Evaluation
2
Mitomycin C-Induced Bacterial Stress Response
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Mitomycin C (Sigma, Sydney, NSW, Australia) was used to induce the putative antibacterial proteins as outlined by [46 (link)]. Single colonies of bacteria were used to inoculate 5 mL of LB (Miller, Sigma) broth, which was placed on an orbital shaker (Conco, TU 4540, Taibei, Taiwan) at 250 rpm and 30 °C overnight. Aliquots (500 µL) of the overnight culture were transferred to further inoculate 25 mL of LB broth. The culture was left to grow (~10–12 h) at 250 rpm and 30 °C until it attained turbidity. Two concentrations (1 µg/mL and 3 µg/mL) of Mitomycin C (Sigma) were independently added into the flasks, which were then positioned on an orbital platform for incubation with rotation at 40 rpm at ambient temperature (24°C). OD600nm readings were recorded through an Ultrospec-10 spectrophotometer (Amersham Biosciences, Amersham, UK) after 2, 4, 6, and 24 h of Mitomycin C (Sigma) addition. The flasks were monitored for signs of cell lysis (clearing of the culture or accumulation of bacterial debris). The flasks without the addition of Mitomycin C served as a control. All the treatments in the experiment were technically replicated four times. OD600nm readings at various time intervals were pooled and statistically analysed using the ANOVA (Analysis of Variance) test through the Genstat 20th edition programme.
3
Mitomycin C-Induced Phage Quantification
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For mitomycin C induction, overnight (∼16-h) cultures were diluted to an OD600 of 0.1, and at an OD600 of 0.3, mitomycin C was added (0.5 μg/ml; Sigma-Aldrich). Samples were harvested every hour postinduction for 5 h to determine the number of CFU and PFU per milliliter. For analysis of the number of PFU per milliliter, cells were centrifuged (21,130 × g for 1 min) and the supernatants were filter sterilized (pore size, 0.22 μm; Millipore). As a lytic host, we used L. reuteri LRΔΦ1ΔΦ2ΔattB1ΔattB2 (VPL4090) (55 (link)), which was prepared as follows: an ∼16-h culture of lytic host was centrifuged at 3,200 × g for 5 min and washed once in an equal volume with phage diluent (16 mM MgSO4 and 20 mM Tris-Cl, pH 7.5, in distilled H2O), followed by resuspension in phage diluent to an OD600 of 2.0. Subsequently, we added 10 mM CaCl2 to the bacterial suspension. We mixed an equal volume of the lytic host suspension and phage samples (200 μl each) in a 15-ml conical tube and incubated the mixture at 37°C for 1 h. We added 3 ml of 0.2% (wt/vol) agarose harboring 10 mM CaCl2, which was gently inverted three times, and poured the mixture onto MRS agar supplemented with 10 mM CaCl2, followed by 15 h of incubation at 37°C.
4
Tumor Cell Migration and Invasion Assays
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To assess tumor cell migration, 21,000 cells were seeded into each chamber of sterile culture inserts (ibidi, Germany), which were placed in 6-well plates. 12 hours after seeding, cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany) in the incubator and insert were removed. Pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02 at the indicated time points and analyzed with the software tool ImageJ to quantify the kinetic of gap closure over time.
To determine tumor cell invasion, FaDu-shKLK6 and FaDu-Mock cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany), trypsinized and 100,000 cells were seeded in matrigel-coated Boyden chamber inserts (BD Bioscience, Germany), which were placed in 12-well plates. After 48 hours, non-invading cells were removed using cotton swaps and remaining cells were fixed for ten minutes in 4 % PFA and stained with Hoechst 33342 (Calbiochem Merck, Germany). Following mounting with Mowiol, five representative pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02, and the amount of invaded cells was counted with the software tool ImageJ.
To determine tumor cell invasion, FaDu-shKLK6 and FaDu-Mock cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany), trypsinized and 100,000 cells were seeded in matrigel-coated Boyden chamber inserts (BD Bioscience, Germany), which were placed in 12-well plates. After 48 hours, non-invading cells were removed using cotton swaps and remaining cells were fixed for ten minutes in 4 % PFA and stained with Hoechst 33342 (Calbiochem Merck, Germany). Following mounting with Mowiol, five representative pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02, and the amount of invaded cells was counted with the software tool ImageJ.
5
Maternal-Paternal Lymphocyte Proliferation
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Splenocytes from five pregnant CBA/J mice from each group on day 14 of gestation were used as responder cells, and paternal splenocytes were used as stimulator cells. Firstly, 100 µl responder cells (2×105 cells/well) and 100 µl mitomycin C (50 µg/ml; Sigma-Aldrich; Merck KGaA)-treated stimulator cells (2×105 cells/well; stimulator cells were incubated with mitomycin C at 37°C for 30 min) were aliquoted into 96-well plates. Responder cells cultured with complete medium alone in 96-well plates were used as the control. After a 3-day incubation at 37°C, 3H-thymidine (20 µCurie/well) was added to the cells and incubated for 6 h at 37°C. The cells were harvested onto glass-fiber paper using a cell harvester, and the count per minute (cpm) was measured using a liquid scintillation counter. The proliferative capacity is presented as the stimulatory index (SI), calculated according to the following equation: SI=(cpm of stimulated cultures-cpm of control cultures)/cpm of control cultures. The experiment was repeated three times.
6
In vitro Wound Healing Assay for MSCs
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Place each of the three MSC types in separate wells of a six-well plate, using both the control cultivation medium and the conditioned medium. After incubating for 24 h, the MSCs reached near confluence. Subsequently, a fixed region of cells was gently scratched using a pipette tip (Axygen, United States). Following this, the cells were subjected to two washes with PBS and then treated with 10 μg/mL mitomycin C (Sigma, United States) for a duration of 3–4 h (Diem et al., 2020 (link)), After the mitomycin C treatment, the cells were replenished with appropriate fresh medium. The migration of cells within the scratched area was observed and recorded at the 24-hour mark using a microscope (Leica Optical, United States).
7
Caulobacter Growth and Drug Sensitivity
8
Generating Mouse Embryonic Stem Cells
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Puromycin-resistant MEF feeder cells (Cell Biolabs, CBA-312) and neomycin-resistant MEF feeder cells (Cell Biolabs, CBA-311) were cultured in DMEM high glucose supplemented with 10% fetal bovine serum and 1% PenStrep at 37°C, then inactivated with 10 µg/ml mitomycin C (Sigma, M4287) for 2 hr for mouse embryonic stem cell culture, as previously described (Hai et al., 2018 (link)). DBA/2J mouse ES cell line AC173/GrsrJ (JAX, 000671C02), was cultured on 0.1% gelatin-coated plates with mitomycin C inactivated MEFs, in ES culture medium (DMEM high glucose with 15% fetal bovine serum, 1% MEM Non-Essential Amino Acids, 1% PenStrep, 0.1 mM 2-mercaptoethanol, 103 unit/ml leukemia inhibitory factor (Millipore Sigma, ESG1107), 1 μM PD0325901 (Sigma, PZ0162) and 3 μM CHIR99021 (Sigma, 361571)), at 37°C. All cell lines were detached with trypsin and frozen with 80% ES culture medium supplemented with 10% DMSO and an additional 10% FBS. Only mouse cell lines used. No STR profiling methods are available to authenticate mouse cell lines. DNA sequencing was used to confirm editing of the DBA/2 allele to the AKR allele of the mouse ES cell line used. Mycoplasm testing was negative.
9
Migration Assay with Mitomycin C
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Uniform vertical streaks were made in the monolayer culture with 200 μL pipette tips. The cells were immediately washed three times with RPMI medium with 10% FBS to remove detached cells. Mitomycin C (10 μg/mL; Sigma‐Aldrich) was added to the medium to inhibit proliferation, so that migration can be effectively followed.3 Mitomycin C is known to inhibit DNA synthesis using forming covalent crosslinks between complementary strands of DNA. This ultimately prevents separation of complementary strands of DNA, thereby inhibiting DNA replication. Cell migration was monitored for 24 hours, and pictures were taken at 0, 18, and 24 hours time points with a digital SPOT camera attached to an inverted Nikon phase contrast microscope (Nikon Inc., Melville, NY).
For wound closure assay in PC3 cells with KD of SOX2, siRNA KD of SOX2 was first performed on PC3 cells as described above. Twenty‐four hours post‐KD, the SOX2 siRNA transfected cells and nontargeting control siRNA transfected cells were replated in six‐well plates, grown overnight at 37°C, and allowed to reach near‐confluent levels. The wound closure assay was then performed as mentioned above. Parallel cultures were used to confirm SOX2 KD as compared with control siRNA‐treated cells
For wound closure assay in PC3 cells with KD of SOX2, siRNA KD of SOX2 was first performed on PC3 cells as described above. Twenty‐four hours post‐KD, the SOX2 siRNA transfected cells and nontargeting control siRNA transfected cells were replated in six‐well plates, grown overnight at 37°C, and allowed to reach near‐confluent levels. The wound closure assay was then performed as mentioned above. Parallel cultures were used to confirm SOX2 KD as compared with control siRNA‐treated cells
10
In Vitro Hematopoietic Stem Cell Culture
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