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Schuele Lab Instructional Protocols

Below is a list of established protocols that the Schuele laboratory utilize in their own cell culture laboratory. 

Protocols

  • Immunostaining for Three Germ Layers with Antibodies against β-III-tubulin, AFP, and SMA
    The purpose of the protocol is to confirm that regenerated iPSC colonies can spontaneously differentiate into three germ layers: ectoderm, endoderm, and mesoderm. By immunostaining the differentiated cells with antibodies against β-III-tubulin, Smooth muscle actin (SMA), and Alpha feto protein (AFP), we can verify their ability to develop into all three germ layers.
  • End Point RT- PCR to Confirm Absence of No Integration of Sendai Virus Vector into the iPSC Genome
    In the Schuele Lab, we have been using replication incompetent Sendai virus reprogramming vectors (Life Technologies, # A13780-01) to safely and effectively derive iPSCs from fibroblasts. Advantage of using Sendai viruses is they are non-integrating and remains in the cytoplasm. Once the newly generated iPSC colonies are established in culture, an endpoint RT-PCR can be performed anytime after passage 10. We collect RNA from the cells to carry out the RT-PCR with primers detecting Sendai virus genome and trans-genes to confirm no-integration.
  • iPSC Differentiation Into 3 Germ Layers by EB Formation
    Each iPSC culture has distinguished characteristics of self-renewal and can undergo differentiation to generate derivatives of the three primary germ layers that potentially can develop into all the cell types present in the body. The goal of this characterization protocol is to form EBs from iPSCs which can spontaneously differentiate into three germ layers – ectoderm, mesoderm, and endoderm.
  • Differentiation of Neural Stem Cells (NSC) from Induced Pluripotent Stem Cells (iPSC)
    The protocol is written based on the article: Sally K. Mak and et al. “Small molecules greatly improve conversion of human-Induced Pluripotent Stem cells to the Neuronal lineage.” Stem Cells International, vol2012, article ID 140427, 12 pages
  • Immunostaining of Induced Pluripotent Stem Cells (iPSC) for Pluripotency Markers Oct4, Sox2, Tra 1-60, and SSEA4
    The purpose of this protocol is to perform immunofluorescent staining of iPSCs culture using antibodies against two transcription factor markers, Oct4 and Sox2, and two surface markers, Tra1-60 and SSEA4 to test their pluripotency. The iPSC cultures should give positive results for all four markers.
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