Stem cell 4 marker immunocytochemistry kit

PBMCs or fibroblasts obtained either from human fetal liver tissues or human adult skin were isolated after collagenase dissociation and reprogrammed into iPSCs with the integration-free based Sendai virus (Cytotune 2.0 kit catalog #A16517 from Life Technologies). Fibroblasts were used at low population doubling (<5) to insure high efficiency of reprogramming. Emerging hiPSC colonies were manually picked and cultured under feeder-free conditions in Essential 8 medium on Geltrex-coated dishes (Life Technologies). hiPSC clones were maintained in Essential 8 Flex medium (Life Technologies) in feeder-free conditions and passaged at least 15 times to increase stable pluripotency. hiPSC generation and characterization were performed in the iPSC cell reprogramming core facility of CHU Sainte-Justine. hiPSC colonies were stained with antibodies for anti-human SSEA-4, Sox2, OCT4, and TRA1-60 followed by incubation with appropriate ALEXA-conjugated secondary antibodies using the pluripotent Stem Cell 4-Marker Immunocytochemistry Kit following the manufacturer's instructions (catalog #A24881 from Life Technologies). Karyotypes were produced by G-banding and analyzed by the CHU Sainte-Justine Cytogenetic Department.

Fibroblasts were first isolated either from human fetal liver tissues or human skin after collagenase dissociation. Single cell fibroblasts cultures were then reprogrammed into hiPSCs using integration‐free Sendai virus (Cytotune 2.0 kit catalog # A16517 from Life Technologies). Fibroblasts were used at low population doubling (between 5 and 10) to increase efficiency of reprogramming. Emerging colonies from transduced cells were manually picked and cultured under feeder‐free conditions in Essential 8 and Essential 8 Flex medium on Geltrex‐coated dishes (Life Technologies). hiPSC clones were passaged at least 15 times to increase stable pluripotency. hiPSC generation and characterization were done in the iPSC‐cell reprogramming core facility of CHU Sainte‐Justine. hiPSC colonies were stained with the antibodies for anti‐human SSEA‐4, Sox2, OCT4, and TRA1‐60 overnight at 4°C using the pluripotent Stem Cell 4‐Marker Immunocytochemistry Kit (catalog # A24881 from Life Technologies), followed by incubation with an ALEXA secondary antibodies for 30 minutes at room temperature. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Karyotypes were produced by G‐banding and analyzed by the CHU Ste‐Justine cytogenetic department.