Patients identified as having pregnancies associated with SCAs were offered prenatal fetal chromosomal karyotyping. All prenatal samples were cultured following standard protocols.21 (link) Amniocytes were cultured with BIO-AMF™-2 medium (Biological Industries, Kibbutz Beit-Haemek, Israel) and Chang Medium® D (Irvine Scientific, Santa Ana, CA, USA). Cord blood cells were cultivated with peripheral blood lymphocyte medium (Xiangya Gene Technology, Hunan, China). At least 20 G-banded metaphases from each sample were analysed using the Wright’s staining method.22 (link)
Chang medium d
Manufactured by Irvine Scientific
Sourced in Israel, United States
Chang Medium® D is a basal medium designed for the culture and maintenance of various cell types, including Chang Conjunctival cells. It provides the essential nutrients and growth factors required to support cell growth and proliferation in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Bio amf 2 medium, by Sartorius (2 mentions)
Alpha mem glutamax, by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Normocin, by InvivoGen (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Glass bottom culture dishes, by MatTek (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Genechip command console software version 4, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
5 protocols using chang medium d
1
Prenatal Fetal Chromosomal Karyotyping
2
Isolation and Culture of Amniotic Fluid Stem Cells
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Collection of amniotic fluid and derived products was approved by the Institutional Review Board at New York University School of Medicine. Amniotic fluid was collected at the time of scheduled, term cesarean delivery with the patient’s informed consent (study# i15-01269, New York, NY, USA) and cultured per published protocols to enrich for AFMSCs [13 (link)]. Briefly, amniotic fluid stem cells were isolated from the fluid and cultured in monolayer in medium consisting of 20% Chang medium D (Irvine Scientific, Irvine, CA, USA), MEM-alpha GlutaMAX (Life Technologies, Carlsbad, CA, USA), 15% embryonic stem cell-qualified fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 100 µg/mL Normocin (InvivoGen, San Diego, CA, USA), then cryopreserved at passage 2. Following thawing, cells were incubated at 37 ℃, 5% CO2, 95% humidity until 80% confluent. We detached cells from the plate using Accutase (Thermo Scientific, Waltham, MA, USA) for further passages or experiments. For the design of this study, AFMSCs from 3 separate batches of amniotic fluid were combined in cell culture.
3
Immunocytochemical Analysis of Lysosomal Proteins
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Fibroblast cells were cultured in Chang Medium D (Irvine Scientific) supplemented with 1% antibiotic (Sigma-Aldrich) and fetal bovine serum (final concentration 20%, Atlanta Biologicals) at 37 °C with 5% CO2.
Cells were seeded in 35-mm-diameter glass bottom culture dishes (MatTek Corporation) and allowed to grow for 24 h at 37 °C in growth medium. For immunocytochemical analysis of LAMP1 and cathepsin D, the cells were then fixed in freshly made 4% paraformaldehyde in warm PBS for 10 min, followed by 5 min post-fix in 100% methanol at −20 °C. After washing in PBS, cells were permeabilized and blocked with PBS containing 0.05% saponin and 5% bovine serum albumin (same solution for diluting antibodies) for 1 h at room temperature. Cells were then incubated with primary antibodies at 4 °C overnight and conjugated secondary antibodies for 2 h at room temperature. After washing, cells were mounted with ProLong Gold antifade reagent with DAPI (Life Technologies) and allowed to cure for 24 h at room temperature in the dark before imaging.
Cells were seeded in 35-mm-diameter glass bottom culture dishes (MatTek Corporation) and allowed to grow for 24 h at 37 °C in growth medium. For immunocytochemical analysis of LAMP1 and cathepsin D, the cells were then fixed in freshly made 4% paraformaldehyde in warm PBS for 10 min, followed by 5 min post-fix in 100% methanol at −20 °C. After washing in PBS, cells were permeabilized and blocked with PBS containing 0.05% saponin and 5% bovine serum albumin (same solution for diluting antibodies) for 1 h at room temperature. Cells were then incubated with primary antibodies at 4 °C overnight and conjugated secondary antibodies for 2 h at room temperature. After washing, cells were mounted with ProLong Gold antifade reagent with DAPI (Life Technologies) and allowed to cure for 24 h at room temperature in the dark before imaging.
4
Isolation of Human Skin Fibroblasts
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Skin biopsies were obtained from patient, Ctrl-1, and Ctrl-2, and incubated in CHANG Medium® D (Irvine Scientific, Santa Ana, CA., USA), with 10% Fetal Bovine Serum (FBS) (Gibco, Thermo Fisher SCIENTIFIC, Waltham, MA, USA). After two weeks, once fibroblasts (FBs) have migrated from the skin fragment and grew in the culture dish, they were isolated using trypsin. In the first three days, fibroblasts were cultivated combining the Chang Medium® D (25%) with the RPMI 1640 medium (75%) (Gibco, Thermo Fisher SCIENTIFIC), supplemented with 10% FBS. Then, Chang medium D was completely replaced by RPMI medium and FBS.
5
Cytogenetic and Microarray Analysis of Amniotic Fluid
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Individuals who showed abnormalities in NIPT received ultrasound‐guided amniocentesis at 18‐24 weeks of pregnancy after informed consent was signed by pregnant women and their families. Amniotic fluid samples were collected from the patients for conventional G‐banded cytogenetic assays and microarray analysis. Amniocytes were isolated and cultured using BIO‐AMF™‐2 medium (Biological Industries, Kibbutz Beit‐Haemek, Israel) and Chang Medium® D (Irvine Scientific, Santa Ana, CA, USA) at 37°C with 5% CO2 for 6‐7 days. The cells at metakinesis were harvested to prepare slides according to the statements in published article.16 Then, G‐band staining was performed according to the Internal System for Human Cytogenomic Nomenclature 2016.
In addition, microarray analysis was also performed for the patients. In brief, genomic DNA was extracted from amniotic fluids using Genomic DNA Extraction kit (QIAamp DNA Blood Mini kit; Qiagen GmBH, Hilden, Germany). DNA samples were digested, ligated and amplified via PCR method. Then, obtained products were processed according to standard procedures of Affymetrix CytoScan 750K Array analysis. The results were processed in Affymetrix GeneChip Command Console software (version 4.0) and Chromosome analysis software (Chromosome Analysis Suite version 2.1) (Affymetrix; Thermo Fisher Scientific, Inc).
In addition, microarray analysis was also performed for the patients. In brief, genomic DNA was extracted from amniotic fluids using Genomic DNA Extraction kit (QIAamp DNA Blood Mini kit; Qiagen GmBH, Hilden, Germany). DNA samples were digested, ligated and amplified via PCR method. Then, obtained products were processed according to standard procedures of Affymetrix CytoScan 750K Array analysis. The results were processed in Affymetrix GeneChip Command Console software (version 4.0) and Chromosome analysis software (Chromosome Analysis Suite version 2.1) (Affymetrix; Thermo Fisher Scientific, Inc).
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