Amniomax c 100 supplement

Manufactured by Thermo Fisher Scientific

Sourced in United States, Belgium

The AmnioMax C-100 Supplement is a cell culture media supplement designed to support the growth and maintenance of human amniotic fluid-derived cells. It provides a defined, serum-free formulation to facilitate cell expansion and differentiation.

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Lab products found in correlation

23 protocols using amniomax c 100 supplement

Isolation and Expansion of Dermal Fibroblasts

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Explants of ~3-mm dermal biopsies were minced and placed in a 100-mm TC-treated tissue culture dish (Corning Life Science, USA) with a small drop of Fibroblast medium [Gibco AmnioMAX-C100 Basal Medium supplemented with 15% AmnioMAX-C100 Supplement (Invitrogen, Carlsbad, CA, USA). Dishes were incubated at 37°C in a humidified 5% CO₂ atmosphere and media was replaced every 3–4 days. Fibroblast outgrowths were harvested by trypsinization, transferred into a new dish with a modified Fibroblast Medium (1:1 solution of Gibco AmnioMAX-C100 Supplemented with 15% AmnioMAX-C100 Supplement (Invitrogen, Carlsbad, CA, USA) and RPMI-1640 Basal Medium supplemented with 10% fetal bovine serum (Invitrogen), 1X Primocin (Invitrogen), 1% non-essential amino acids (Invitrogen), with media exchange every 3–4 days and allowed to reach 90% confluence prior to splitting. Fibroblast cultures were passaged no more than 3 times prior to collection for DNA, RNA, or protein extracts isolation or cryopreservation.

Pretto D., Yrigollen C.M., Tang H.T., Williamson J., Espinal G., Iwahashi C.K., Durbin-Johnson B., Hagerman R.J., Hagerman P.J, & Tassone F. (2014). Clinical and molecular implications of mosaicism in FMR1 full mutations. Frontiers in Genetics, 5, 318.

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Skin Biopsies of Patients with CHM Mutations

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Skin biopsies of patients carrying a deletion of the CHM gene, a UAA nonsense mutation and a UGA nonsense mutation were performed following informed consent at the Centre of Reference for Genetic Sensory Disorders (CHRU Montpellier, France). The biopsy of the patient carrying a deletion of exon 8 performed at the same centre was previously reported [9 (link)]. The skin biopsy of the patient carrying a UAG nonsense mutation was performed at the IIS-Fundacion Jimenez Diaz (Madrid, Spain). The skin biopsies and emerging fibroblasts were cultured in AmnioMAX C100 basal media with L-glutamine (Invitrogen, Life Technologies, Saint Aubin, France) containing 10% decomplemented FCS (Lonza, Verviers, Belgium), 1% penicillin-streptomycin-amphotericin B (Lonza) and 2% AmnioMax-C100 supplement (Invitrogen, Life Technologies) at 37°C under 5% CO2 as described [9 (link)]. This work was performed under the biomedical research authorization number 2014-A00549-38.

Sanchez-Alcudia R., Garcia-Hoyos M., Lopez-Martinez M.A., Sanchez-Bolivar N., Zurita O., Gimenez A., Villaverde C., Rodrigues-Jacy da Silva L., Corton M., Perez-Carro R., Torriano S., Kalatzis V., Rivolta C., Avila-Fernandez A., Lorda I., Trujillo-Tiebas M.J., Garcia-Sandoval B., Lopez-Molina M.I., Blanco-Kelly F., Riveiro-Alvarez R, & Ayuso C. (2016). A Comprehensive Analysis of Choroideremia: From Genetic Characterization to Clinical Practice. PLoS ONE, 11(4), e0151943.

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Human Fibroblast Cell Culture Protocol

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The following method for human fibroblast cell culturing was originally described in our previous report [21 (link)]. For the siRNA transfection to knock down the Sam68, an established human fibroblast cell line was placed in treated tissue culture T-25 flask (Corning Life Science, USA) with a modified Fibroblast Growth Medium [1:1 solution of Gibco AmnioMAX-C100 supplemented with 15% AmnioMAX-C100 Supplement (Invitrogen, Carlsbad, CA, USA) and RPMI-1640 Basal Medium supplemented with 10% fetal bovine serum (Invitrogen), 1X primocin (Invitrogen), 1% non-essential amino acids (Invitrogen), and 1:250 fungizone (J R Scientific, Woodland, CA, USA)] with media exchange every 5 days and allowed to reach 90% confluence prior to splitting. Fibroblast cultures were passaged no more than 3 times prior to transfection.

Zafarullah M., Li J., Tseng E, & Tassone F. (2023). Structure and Alternative Splicing of the Antisense FMR1 (ASFMR1) Gene. Molecular neurobiology, 60(4), 2051-2061.

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Choroideremia Skin Biopsy and Fibroblast Culture

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A skin biopsy on the inner side of the upper arm of a 16-year-old boy (referred to as CHM1) with a confirmed molecular diagnosis of choroideremia (deletion of exon 8 of CHM identified by RT-PCR; Klinkum der Universität, Regensburg, Germany) was performed under sterile conditions at the Centre of Reference for Genetic Sensory Disorders (CHRU Montpellier) following informed consent. The Ministry of Public Health accorded approval for biomedical research under the authorization number 11018S. The skin biopsy specimen was rinsed in PBS, cut into small pieces, and cultured in 35-mm culture dishes (two pieces per dish) in AmnioMax-C100 basal media with l-glutamine (Invitrogen, Life Technologies, St Aubin, France) containing 10% decomplemented fetal calf serum (Lonza, Verviers, Belgium), 1% penicillin–streptomycin–amphotericin B (Lonza), and 2% AmnioMax-C100 supplement (Invitrogen, Life Technologies) at 37 °C under 5% CO₂. The biopsies were removed to a fresh dish once the emerging fibroblasts reached 80% confluence. The cells were passaged, and aliquots ranging from P1 to P5 from each culture were frozen in fetal calf serum containing 10% dimethyl sulfoxide (Sigma Aldrich, St Quentin Fallavier, France).

Cereso N., Pequignot M.O., Robert L., Becker F., De Luca V., Nabholz N., Rigau V., De Vos J., Hamel C.P, & Kalatzis V. (2014). Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient. Molecular Therapy. Methods & Clinical Development, 1, 14011-.

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Dermal Fibroblast Culture Protocol

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Explants of dermal biopsies (~ 3 mm) were minced and placed in a 100-mm TC-treated tissue culture dish (Corning Life Science) with 5 mL fibroblast medium (Gibco AmnioMax-C100 Basal Medium with 15% AmnioMax-C100 Supplement (Invitrogen)) and placed in 37°C humidity incubators with 5% CO₂ atmosphere. Media was replaced every 3–4 days until fibroblast outgrowths from one explant were a quarter of the size of the dish. Fibroblasts were passaged into a new dish by trypsinization, and grown in modified fibroblast medium (1 part AmnioMax-C100 medium (as described above), 1 part RPMI-1640 medium (RPMI-1640 basal medium (Invitrogen) supplemented with 1X Primocin (Invivogen), 1% non-essential amino acids, and 10% fetal bovine serum (Corning Life Science)). Media was replaced every 3–4 days and allowed to reach 90% confluence prior to passaging. Fibroblast cultures were passaged between 2–20 times prior to collection for DNA, RNA, cryopreservation, or cell activation.

Yrigollen C.M., Pacini L., Nobile V., Lozano R., Hagerman R.J., Bagni C, & Tassone F. (2016). Clinical and Molecular Assessment in a Female with Fragile X Syndrome and Tuberous Sclerosis. Journal of genetic disorders & genetic reports, 5(3), 139.

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Signaling Pathway Analysis in NSML Fibroblasts

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Skin fibroblasts isolated from the NSML patient and a normal (wild type) donor of the same age were cultured in AmnioMax C-100 medium (Invitrogen) with AmnioMax C-100 supplement (Invitrogen). Cells were starved overnight in medium without supplement and then were either left unstimulated or were stimulated with 10 nmol/l insulin-like growth factor (IGF) or 25 ng/ml epidermal growth factor (EGF) for the indicated times. After protein extraction, immunoblots were performed, following the manufacturer’s directions, with anti-phospho-AKT, anti-phospho-ERK1/2, anti-phospho-S6RP, and their respective total proteins (Cell Signaling). Bands were visualized with enhanced chemiluminescence and quantified by densitometry using ImageJ 1.41 software (Wayne Rasband, http://rsbweb.nih.gov/ij/). Protein expression was referred to the total amount of respective proteins. All analyses were performed 4-5 times. Data are presented as mean ± SEM. Statistical significance was determined using 1-way ANOVA or 2-way repeated measure ANOVA, as appropriate. If ANOVA was significant, individual differences were evaluated using the Bonferroni post-test. Values of P < 0.05 were considered statistically significant.

Hahn A., Lauriol J., Thul J., Behnke-Hall K., Logeswaran T., Schänzer A., Böğürcü N., Garvalov B.K., Zenker M., Gelb B.D., von Gerlach S., Kandolf R., Kontaridis M.I, & Schranz D. (2015). Rapidly Progressive Hypertrophic Cardiomyopathy in an Infant with Noonan syndrome with multiple Lentigines. Palliative Treatment with a Rapamycin Analog. American journal of medical genetics. Part A, 0(4), 744-751.

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Skin Biopsy and Fibroblast Culture

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The skin biopsy of a 10 year-old boy (CHM3) carrying a UAA nonsense mutation was performed at the Centre of Reference for Genetic Sensory Disorders (CHRU Montpellier, France) according to local guidelines and regulations. Informed consent was obtained from the subject’s mother. All experimental protocols were approved by the National Agency for Health and Medicine under the biomedical research authorisation number 2014-A00549-38. The corresponding fibroblasts were cultured in AmnioMAX C100 basal media with GlutaMAX (Gibco, ThermoFisher Scientific, Villebon sur Yvette, France) containing 10% decomplemented foetal calf serum (FCS; Lonza, Verviers, Belgium), 1% penicillin-streptomycin-amphotericin B (Lonza) and 2% AmnioMax-C100 supplement (Gibco) at 37 °C under 5% CO₂^{30 (link)}.

Torriano S., Erkilic N., Baux D., Cereso N., De Luca V., Meunier I., Moosajee M., Roux A.F., Hamel C.P, & Kalatzis V. (2018). The effect of PTC124 on choroideremia fibroblasts and iPSC-derived RPE raises considerations for therapy. Scientific Reports, 8, 8234.

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Skin Biopsy and Fibroblast Culture of NR2E3-associated adRP

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The skin biopsy of the NR2E3-associated adRP patient volunteer was performed at the National Reference Center for Inherited Sensory Diseases (Maolya, Montpellier, France) following signed informed consent. The biomedical research study (project identification code 2014-A00549-38) was approved on the 13 June 2014 under the authorization number 140549B-62 by the French National Agency for the Safety of Medicines and Health Products (ANSM). The biopsy and emerging fibroblasts were cultured in AmnioMAX C100 basal media with L-glutamine (Gibco, Thermo Fisher Scientific, Villebon sur Yvette, France) that contained 10% decomplemented foetal bovine serum (FBS; Gibco), 1% penicillin-streptomycin amphotericin B (Gibco), and 2% AmnioMax-C100 supplement (Gibco). The fibroblasts were passaged using 0.25%Trypsin (Gibco) and then cryo-preserved in FBS that was supplemented with 10% DMSO (Sigma Aldrich, St. Quentin Fallavier, France).

Diakatou M., Dubois G., Erkilic N., Sanjurjo-Soriano C., Meunier I, & Kalatzis V. (2021). Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3. International Journal of Molecular Sciences, 22(5), 2607.

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Calcium Signaling in Renal Cell Carcinoma

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Human renal cell carcinoma cell line 768-O was obtained from LGC Promochem (Wesel, Germany). Cells were cultured in AmnioMAX C100 Basal Medium supplemented with AmnioMAX C-100 Supplement (Gibco). The cell line was incubated in a moistened atmosphere at 5% CO₂ at 37° C. Every time cells were passaged they were washed with sterile DPBS, and detached by using 2.5% trypsin-EDTA. Cells were centrifuged at 300 g for five minutes and resuspended in culture medium. For the experiments, cells were treated with calcium alone (5 mM, 30 min, serum-free) or alternatively with the CaSR antagonist NPS2143 (10 µM, 30 min, serum-free) and afterwards with the combination of NPS2143 (10 µM) and calcium (5 mM) for further 30 min. Serum-free medium served as a control.

Frees S., Breuksch I., Haber T., Bauer H.K., Chavez-Munoz C., Raven P., Moskalev I., D´Costa N., Tan Z., Daugaard M., Thüroff J.W., Haferkamp A., Prawitt D., So A, & Brenner W. (2018). Calcium-sensing receptor (CaSR) promotes development of bone metastasis in renal cell carcinoma. Oncotarget, 9(21), 15766-15779.

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Skin Biopsies of USH2A Volunteers

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Skin biopsies of the USH2A volunteers were performed under sterile conditions at the Center of Reference for Inherited Sensory Diseases (CHU Montpellier) following informed consent as part of the registered clinical trial NCT03853252. Ethics approval was provided by the local institutional review board and the French National Agency for the Safety of Medicines and Health Products (ID 2014-A00549-38). The biopsies and emerging fibroblasts were cultured in AmnioMAX C100 basal medium supplemented with GlutaMAX (Gibco), 10% decomplemented fetal calf serum (FCS; Gibco), 1% penicillin-streptomycin-amphotericin B (Gibco), and 2% AmnioMax C100 supplement (Gibco), as described previously.³⁸ (link)

Sanjurjo-Soriano C., Jimenez-Medina C., Erkilic N., Cappellino L., Lefevre A., Nagel-Wolfrum K., Wolfrum U., Van Wijk E., Roux A.F., Meunier I, & Kalatzis V. (2023). USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids. Human Genetics and Genomics Advances, 4(4), 100229.

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