For collagen gel substrate, collagen solutions were prepared by mixing solution of 8:1:1 Cellmatrix I-A (Nitta Gelatin Inc., Osaka, Japan)/Minimum essential medium (MEM) (10X) (Life Technologies, Tokyo, Japan) without NaHCO3/sterile reconstitution buffer (22 mg/mL of NaHCO3, 0.005 M of NaOH, and 200 mM of HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-sulfonic acid; Gibco, Life Technologies), yielding a homogeneous solution at 0 °C. Then, the 30 µL of collagen solution was added onto 96-well plates and heated at 37 °C for 30 min. Collagen-coated substrate were prepared by adding Cellmatrix I-C (Nitta Gelatin Inc.) solution onto 96-well plates and heated at 37 °C for 2 h. Then added solution was removed.
Cellmatrix 1 c
Manufactured by Nitta Gelatin
Sourced in Japan
Cellmatrix I-C is a laboratory product developed by Nitta Gelatin. It is a type I collagen-based matrix that can be used for cell culture and tissue engineering applications. The core function of Cellmatrix I-C is to provide a three-dimensional scaffold for the growth and proliferation of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cellmatrix 1 a, by Nitta Gelatin (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Dmem ham s f 12, by Nacalai Tesque (1 mentions)
Earle s balanced salt solution, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
70 μm cell strainer, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Deoxyribonuclease 1, by Roche (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Cell matrix 1 p, by Nitta Gelatin (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
A549 human lung adenocarcinoma cell line, by American Type Culture Collection (1 mentions)
6 protocols using cellmatrix 1 c
1
Collagen Gel Substrate Preparation
2
Cell Migration Monitoring in 2D and 3D
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For microscopic observation of random migration, cells (7 × 102) were plated onto multi-well glass-bottom dishes (Matsunami glass) coated with 10 μg/ml fibronectin (Sigma) or acidified type-I collagen (Cellmatrix I-C, Nitta Gelatin) and containing 200 μl Leibovitz's L-15 medium (Gibco) containing 10% FBS. After a pre-incubation period, the cells in 5 fields were time-lapse recorded for 4-8 h as described previously (Morioka et al., 2009). The migration of 5 well-isolated, non-dividing cells per field was tracked on a monitor, and their speed and directional persistence were calculated from a time series of coordinates using Dunn's formula23 (link). For 3D-culture in collagen gel, cells suspended in 0.25 ml DME containing 2.4 mg/ml type I collagen (Cellmatrix I-A, Nitta Gelatin) freshly reconstituted on ice were layered onto a basal layer (0.25 ml, the same composition as top layer except the cells) preset in 12-well plates, and the plates were incubated in a CO2 incubator at 37°C. The gel was overlaid with 1 ml growth medium after 30 min.
3
Isolation of Sertoli Cells from Postnatal Testes
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Testes at postnatal days 1 and 21 (P1 and P21) were incubated in Earle’s balanced salt solution (Sigma, St. Louis, MO, USA) containing 1 mg/ml collagenase (Thermo Fisher Scientific), 0.5 mg/ml dispase (Thermo Fisher Scientific) and 2.5 mg/ml trypsin (Sigma) at 34 °C for 30 min, and following the addition of 0.3 mg/ml deoxyribonuclease I (Roche, Basel, Switzerland), were incubated for another 30 min at 34 °C. After centrifugation, the cells were suspended in PBS with 7-amino-actinomycin D (7AAD; BD Bioscience, Franklin Lakes, NJ, USA). The cells were washed with PBS containing 0.3 mg/ml deoxyribonuclease I, and filtered using a 70 μm cell strainer (BD Bioscience). EGFP-positive Sertoli cells were isolated by fluorescence-activated cell sorting (FACS) using a JSAN cell sorter (Bay bioscience, Kobe, Japan). For siRNA treatment, Sertoli cells at P21 were cultured at 34 °C on 24-well culture plates (Asahi Glass, Tokyo, Japan) pre-coated with collagen type I (Cell Matrix I-C; Nitta Gelatin, Osaka, Japan) in DMEM/Ham’s F-12 (1:1; Nacalai Tesque) supplemented with 10% FBS and Penicillin-streptomycin-glutamine (Thermo Fisher Scientific).
4
Establishing 2D Cell Line from CTOS
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The Src-specific RNA interference (RNAi) and control RNAi were from the siGENOME Human siRNA-SMART pool (Thermo Scientific Dharmacon, Lafayette, CO). C45-2D cells were established from the C45 CTOS line. Briefly, C45 CTOS were cultured on a type I collagen (Cellmatrix I-C, Nitta Gelatin)ecoated dish with DMEM/F12 (Invitrogen) and 10% fetal bovine serum. The CTOSs that grew on the dish were passaged once each week. After several passages, C45-2D cells were established. RNAi was transfected to C45-2D cells by lipofection using Lipofectamine RNAiMAX (Life Technologies).
5
Establishment of Irradiation-Tolerant A549 Cells
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The A549 human lung adenocarcinoma cell line was purchased from American Type Culture Collection (Manassas, VA). Subclonal parental A549 cells (P-3 cells) and irradiation-tolerant P-3 cells (IR cells) were established as we previously reported (Ishihara et al., 2010) . The cells were cultured with Dulbecco's modified Eagle medium (Sigma-Aldrich, St. Lois, MO) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France) and 1% antibiotic/antimycotic solution (Sigma-Aldrich). The cells were incubated in a humidified incubator with 5% CO 2 at 37°C. A type I collagen solution (0.3 mg/mL; Cell matrix I-C; Nitta Gelatin, Osaka, Japan) was used to prepare a collagen-coated glass dish (Corning, Tewksbury, MA). A type I collagen solution (0.9 mg/mL; Cell matrix I-P; Nitta Gelatin) was used for making a collagen gel.
6
Time-lapse Imaging of Mitotic Cells
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Transfected cells were plated onto glass-bottom dishes (Iwaki, Tokyo, Japan) coated with 0.3 mg/mL type-I collagen (Cell matrix I-C; Nitta Gelatin). Two-dimensional time-lapse images were acquired using an inverted microscope (IX71; Olympus) equipped with a single-chip color CCD camera (DP70; Olympus) and an objective lens (UPlanFl 100´/1.30 NA Oil; Olympus). During observation, cells were warmed on a thermoplate set to 37°C (MATS-U55R30; Tokai Hit, Fujinomiya, Japan). Images of rounded cells were acquired every 30 s from anaphase onset using DP Controller software (Olympus).
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