Protocols - Creative Biogene

Protocol

3D Tumorsphere Culture Protocol for Cancer Research

Experiment Tags: 3D Tumorsphere Culture, Cancer Research, Matrigel Embedding, Tumor Tissue Culture, Cell Transduction, Cryopreservation, Cell Counting

Experiment Summary: This protocol outlines a comprehensive procedure for 3D tumorsphere cultivation, including cell transduction, 3D tumorsphere culture using Matrigel, primary tumorsphere culture from fresh tumor tissue, spheroid passaging, and cryopreservation. The protocol aims to replicate the 3D environment of solid tumors, aiding researchers in studying tumor biology and conducting drug screening.

Protocol

CRISPR/Cas9-Mediated Gene Editing in C. elegans

Experiment Tags: CRISPR-Cas9, Gene Editing, C. elegans, sgRNA, Plasmid Construction Homologous Recombination, Genetic Screening.

Experiment Summary: This protocol outlines the process for performing CRISPR/Cas9-mediated genome modifications in C. elegans, including the design of specific sgRNA sequences, construction of Cas9-sgRNA plasmids, and preparation of injection mixtures.

Protocol

Dual-Fluorescence Flow Cytometry for Protein Level Analysis

Experiment Tags: Dual-fluorescence, Flow Cytometry, Protein Analysis, Steady-State Protein Levels, eGFP mCherry, Transfection Efficiency, Cell Line Transfection, Protein Abundance

Experiment Summary: This protocol provides a powerful tool for studying protein expression levels under various cellular conditions, offering single-cell resolution and the ability to detect subtle differences that might be masked in bulk assays. Its versatility makes it applicable to a wide range of proteins and cellular contexts, contributing to our understanding of protein homeostasis and cellular biology.

Protocol

Intraductal Injection of Adenoviral Cre for Temporally Controlled Breast Cancer Modeling in Mice

Experiment Tags: Breast Cancer, Mouse Model, Intraductal Injection, Adenoviral Cre, Temporal Control

Experiment Summary: This protocol outlines a technique for injecting adenoviral Cre (Ad-Cre) intraductally into mice mammary glands to create animal models of breast cancer. By employing cell-type-specific promoters to target mammary epithelial cells (MECs), this strategy enables fine temporal control of cancer induction and cell-type specificity. The process is described in the protocol, which includes creating and caring for floxed mice, monitoring the growth of breast tumors, and providing postoperative care.

Protocol

Monkeypox Antigen Expression and Antibody Response Protocol

Experiment Tags: MPXV, A29L protein, B6R protein, M1R protein, HEK293T cells, Recombinant proteins, Protein purification, SDS-PAGE, Western blot, ELISA, Neutralization assay, Antibody titer

Experiment Summary: This protocol outlines the process for expressing, purifying, and analyzing MPXV antigens and assessing their immunogenicity, which is vital for vaccine research and development.

Protocol

Protocol for Cell Viability Assays: CCK-8 and MTT

Experiment Tags: CCK-8 assay, MTT assay, cell viability, drug treatment, IC50, absorbance measurement, cell seeding, aseptic technique, OD values, 96-well plate, biological assay, cytotoxicity, experimental protocol.

Experiment Summary: This protocol provides a step-by-step guide for performing CCK-8 and MTT cell viability assays, covering cell seeding, drug concentration settings, treatment duration, and absorbance measurement. It also includes important considerations for avoiding contamination, ensuring cell health, and interpreting results accurately, especially in high-throughput drug testing.

Protocol

Protocol for CRISPR/Cas9-mediated Knockout in Difficult-to-Transfect Cells

Experiment Tags: CRISPR/Cas9, Lentivirus, Gene Knockout, Cell Line Development

Experiment Summary: The protocol utilizes lentiviral vectors and a control system to regulate Cas9 activity, enabling the development of knockout cell lines. The development of a human muscle cell line that targets the gene serves as evidence of the protocol's effectiveness.

Protocol

Protocol for Mitochondria Transfer between Mesenchymal Stem Cells and Glioblastoma Stem Cells

Experiment Tags: Mitochondria Transfer, Mesenchymal Stem Cells, Glioblastoma Stem Cells, Cell Interaction, Cell Biology.

Experiment Summary: This protocol facilitates the transfer of mitochondria from mesenchymal stem cells (MSCs) to glioblastoma stem cells (GSCs). This protocol allows for the investigation of the effects of mitochondria on GSC metabolism, proliferation, and response to therapy by isolating mitochondria from MSCs and transferring them to GSCs.

Protocol

Protocol for rAAVrh.74-SaCas9/gRNA Vector-Mediated Postnatal Cardiac Gene Editing in mdx/utrophin+/- Mice

Experiment Tags: CRISPR, Genome Engineering, Cardiac Gene Editing, rAAV Delivery, Dystrophin Restoration

Experiment Summary: This protocol describes how to use the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system to perform postnatal cardiac gene editing in mdx/utrophin+/- mice. Specifically, the protocol uses the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and guide RNAs (gRNAs) delivered via recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) to carry out the gene editing. Restoring dystrophin expression in these mice's cardiac muscles is the main goal in hopes of developing a treatment for Duchenne muscular dystrophy (DMD).

Protocol

Rapid and Efficient Gene Editing in Human Pluripotent Stem Cells via FLPe Recombinase-Mediated Cassette Exchange at the AAVS1 Locus

Experiment Tags: Gene Editing, FLPe Recombinase, Cassette Exchange, AAVS1 Locus, Human Pluripotent Stem Cells

Experiment Summary: The protocol presents a unique approach to FLPe recombinase-mediated cassette exchange (RMCE) gene editing at the AAVS1 locus in human pluripotent stem cells (hPSCs) that is both rapid and efficient. This method makes it easier to generate highly reliable isogenic lines, which in turn makes transgenesis studies in hPSCs easier to conduct.