CD4+ T cells were isolated from the spleens of BALB/c or transgenic mice by negative selection using lab grown antibodys (Ab) to MHC class II I-Ad (clone 212.A1), CD8 (clone TIB 105), B220 (clone TIB 164), and FcR (clone 24G2) followed by anti-Ig-coated magnetic beads (Polysciences). Syngeneic T cell-depleted splenocytes were used as antigen presenting cells (APC) and were prepared by Ab-mediated rabbit complement lysis using Abs to CD4 (clone GK1.5) and CD8 (clone TIB 105) followed by mitomycin C treatment (Sigma-Aldrich) [13] , [14] (link). 0.5×106/ml CD4+ T cells and 1.0×106/ml APCs were cultured with 5 µg/ml pOVA323–339 and (i) with 10 U/ml recombinant murine IL-2 (Roche), 10 ng/ml recombinant murine IL-4 (PeproTech), and anti-IFN-gamma (XMG1.2), or (ii) with 25 U/ml recombinant murine IL-2 (Roche), 5 ng/ml recombinant murine IL-12 (Peprotech) and anti-IL-4 (11B11), or (iii) with 20 ng/ml recombinant murine IL-23 (eBioscience), 2 ng/ml recombinant human TGFbeta (Peprotech), 40 ng/ml recombinant murine IL-6 (Miltenyi Biotec), anti-IL-4 (11B11), anti-IL-2 (JES6-1A12) and anti-IFN-gamma (XMG1.2) for 4–7 days to generate (i) Th2 cells, (ii) Th1 cells or (iii) Th17 cells. DO11.10 Th2 purity before transfer ranged between 92–98% as verified by CD4+KJ1-26+ staining by flow cytometry.
Mitomycin c treatment
Manufactured by Merck Group
Sourced in United States
Mitomycin-C is a chemotherapy medication used in the treatment of various types of cancer. It is a type of antibiotic that works by interfering with the growth and division of cancer cells. Mitomycin-C is typically administered intravenously or topically, depending on the specific medical condition being treated.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Recombinant murine il 4, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Miltenyi Biotec (1 mentions)
Recombinant murine il 23, by Thermo Fisher Scientific (1 mentions)
Recombinant murine il 12, by Thermo Fisher Scientific (1 mentions)
Recombinant murine il 2, by Roche (1 mentions)
Recombinant murine il 6, by Miltenyi Biotec (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Ab63797, by Abcam (1 mentions)
Ab90522, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Influx cytometer, by BD (1 mentions)
C8052, by Merck Group (1 mentions)
M4287, by Merck Group (1 mentions)
A1345, by Merck Group (1 mentions)
P1585, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
7 protocols using mitomycin c treatment
1
Isolation and Polarization of CD4+ T Cells
2
Scratch Assay for LN LEC Migration
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LN LECs were seeded on 96-well plates and incubated until a confluent layer was reached, followed by mitomycin-C treatment (2 µg/mL, Sigma-Aldrich) for 2 h. Thereafter, a scratch was inflicted in each well using a scratch brush (V&P Scientific, San Diego, CA, USA). The cells were washed and 10 µg/mL blocking antibodies against CD200, tenascin, or BST2, or the corresponding isotype controls in either starvation or full LN LEC medium were added. Images were taken with an Axiovert 200M microscope and AxioCam MRm (Carl Zeiss) with 10× magnification at 0 h and 16 h. Scratch closure was quantified using TScratch [44 (link)].
3
Bacterial Immunoblot Analysis Protocol
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For immunoblot assays bacterial strains were grown as previously but with mitomycin-C treatment (5 μg/ml, Sigma) for 4 h at 30 ° C, 120 rpm. Cells were harvested in late log phase, washed twice in PBS, before being centrifuged and subjected to a freeze-thaw cycle. Cells were re-suspended in bacterial lysis buffer (50 mM Tris pH 8.0, 10% v/v glycerol, 0.1% Triton X-100, 100 μg/ml lysozyme, 3 U/ml DNAseI, 2 mM MgCl2, cOmpleteTM Mini (EDTA-free Protease Inhibitor Cocktail [Sigma]), before being sonicated three times at 10 mAmps for 30 s, on ice. Cells were lysed by five passages through a syringe using a 26-gauge needle before centrifugation at 16,000 × g. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific) before Western blotting was carried out using antibodies against either RecA (Abcam, ab63797) or GroEL (Abcam, ab90522).
4
Isolation and Culture of Primary Epidermal Cells
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Primary cells were isolated from whole epidermises of 2 days old WT, CRTC1 KO, CRTC2 KO and CRTC3 KO pups and cultured in RPMI media containing TPA (P1585, Sigma) and cholera toxin (C8052, Sigma), following previously described detailed protocol (Godwin et al., 2014 (link)). For skin melanoblast quantification, WT and CRTC3 KO mice were crossed with mice carrying iDCT:GFP transgene system. Pregnant females were given 1mg/ml doxycycline in water and epidermal cell suspensions were isolated from 2 days old pups (WT N = 5, KO N = 4). GFP positive cells were counted through fluorescence-activated cell sorting (Becton Dickinson Influx cytometer). To account for differences in total amounts of cells isolated from each animal, quantification was expressed as percentage of GFP positive cells per epidermis. In experiments where XB2 feeder keratinocytes were used, cells were obtained from Wellcome Trust Functional Genomics Cell Bank and prepared using mitomycin c treatment (M4287, Sigma), as previously described (Godwin et al., 2014 (link)). In some experiments, catalase (LS001896, Worthington Biochemical) and an antioxidant supplement (A1345, Sigma) were added to the primary cultures in the attempt to improve survival and differentiation of CRTC3 KO melanoblasts.
5
Culturing Mouse Embryonic Fibroblasts and LHSCC Cells
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Mouse embryonic fibroblast cell line 3T3-J2 (RRID:CVCL_W667 ; purchased from Otwo Biotech, Shenzhen, China) was cultured in complete DMEM with high glucose supplemented with 10% (v/v) FBS (Life Technologies) and 100 IU/ml penicillin, and 100 mg/ml streptomycin. In the CR system, 3T3-J2 were mitotically inactivated either by irradiation or by mitomycin C-treatment (2.5 h, 4 mg/ml final concentration, Sigma-Aldrich). Primary LHSCC cells were cultured in Complete F medium (Table 1 ) at 37°C in a 5% CO2 humidified incubator. The medium was renewed every 2 days. The cell numbers of every passage were checked by a cell counter plate.
6
Maintenance of iPSCs and MEFs in Cell Culture
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Cell cultures were maintained at 37 °C in a 5% CO2 humidified incubator. The iPSCs were cultured on hES-qualified Matrigel-coated plates (Corning) in mTeSR1 medium (STEMCELL Technologies) while MEFs in Dulbecco’s modified Eagle medium (DMEM; Gibco) containing 10% foetal bovine serum (FBS, Gibco) and 0.1 mM non-essential amino acids (NEAA; Gibco). MEFs were mitotically inactivated through mitomycin-c treatment (Sigma-Aldrich) 48 h before iPSCs seeding.
7
Culturing and Maintaining Mouse Fibroblasts
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Mouse embryonic broblast cell line 3T3-J2 (RRID: CVCL_W667; purchased from Otwo Biotech, Shenzhen, China) was cultured in complete DMEM with high glucose supplemented with 10% (v/v) FBS (Life Technologies) and 100 IU/ml penicillin, and 100 mg/ml streptomycin. In the CR system, 3T3-J2 were mitotically inactivated either by irradiation or by mitomycin C-treatment (2.5 h, 4mg/ml nal concentration, Sigma-Aldrich). Primary LHSCC cells were cultured in Complete F medium (Table 1) at 37℃ in a 5% CO2 humidi ed incubator. The medium was renewed every two days. The cell numbers of every passage were checked by a cell counter plate.
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