Protocols
These protocols have been created by members of the Fleischman lab and are publicly available. If you use our protocols, please cite us in your publications and presentations. If you have any questions, please contact us.
General Protocols
- Bone Marrow Transduction-Transplantation
- The transduction-transplantation method uses retrovirus expressing a gene of interest, such as CALR, JAK2, or MPL, to initiate an MPN-like disease, with symptoms of either ET or PV and progression to MF. This protocol is supplemented by our bone marrow isolation and viral production protocols.
- The transduction-transplantation method uses retrovirus expressing a gene of interest, such as CALR, JAK2, or MPL, to initiate an MPN-like disease, with symptoms of either ET or PV and progression to MF. This protocol is supplemented by our bone marrow isolation and viral production protocols.
- Isolation of Bone Marrow from the Major Leg Bones
- This protocol describes the process of harvesting bone marrow from the major leg bones.
- This protocol describes the process of harvesting bone marrow from the major leg bones.
- PBMC Isolation from Peripheral Blood
- This protocol details the many facets of blood cell isolation from human peripheral blood, and can easily be applied to bone marrow. Here we have described the isolation of plasma, granulocytes, mononuclear cells (MNCs), and specific subpopulations via magnetic-activated cell sorting (MACS).
- Heme Biorepository Protocol
Cell Culture Protocols
- CFC Assay (Methylcellulose)
- This protocol outlines the general colony-forming cell (CFC) assay using methylcelulose. The specific methylcellulose formulation can be altered to investigate the effects of drugs and/or cytokines on colony formation.
- This protocol outlines the general colony-forming cell (CFC) assay using methylcelulose. The specific methylcellulose formulation can be altered to investigate the effects of drugs and/or cytokines on colony formation.
- Hematopoietic Stem Cell Differentiation Assay
- Plating stem or progenitor cells onto OP-9 stromal cells allows us to examine how certain genes or mutations can effect lineage decisions during hematopoiesis. This protocol is supplemented by our bone marrow isolation and cell surface staining protocols.
- Plating stem or progenitor cells onto OP-9 stromal cells allows us to examine how certain genes or mutations can effect lineage decisions during hematopoiesis. This protocol is supplemented by our bone marrow isolation and cell surface staining protocols.
- Macrophage Preparation and Phagocytosis Assay
- This is a simple macrophage preparation protocol that includes one example of a common phagocytosis assay. This protocol uses fluorescently-labeled targets such as latex beads or apoptotic cells to label phagocytic macrophages. These macrophages can therefore be easily visualized via either flow cytometry or fluorescence microscopy with minimal adjustments. This protocol is supplemented by our PBMC isolation and CFSE staining protocols.
- This is a simple macrophage preparation protocol that includes one example of a common phagocytosis assay. This protocol uses fluorescently-labeled targets such as latex beads or apoptotic cells to label phagocytic macrophages. These macrophages can therefore be easily visualized via either flow cytometry or fluorescence microscopy with minimal adjustments. This protocol is supplemented by our PBMC isolation and CFSE staining protocols.
- WEHI-Conditioned Media Prepration
- The WEHI cell line produces murine IL-3, providing an alternative to recombinant IL-3. WEHI-CM is frequently used as a supplement in liquid and semi-solid media.
- The WEHI cell line produces murine IL-3, providing an alternative to recombinant IL-3. WEHI-CM is frequently used as a supplement in liquid and semi-solid media.
- Viral Production
- This protocol utilizes the X-tremeGENE 9 reagent and 293T cells to produce retrovirus.
Cell Staining Protocols
- Cell Surface Staining and Annexin V Staining
- This protocol describes both general cell surface staining as well as an optional Annexin V staining step. The cell surface staining protocol can be adapted to any number of fluorophores and may be followed by a number of intracellular stains if so desired. One such stain, Annexin V is used as a marker of apoptosis in cells and is typically used in combination with a marker of cell death, in our case PI. This allows healthy, apoptotic, and necrotic cells to be distinguished from one another.
- This protocol describes both general cell surface staining as well as an optional Annexin V staining step. The cell surface staining protocol can be adapted to any number of fluorophores and may be followed by a number of intracellular stains if so desired. One such stain, Annexin V is used as a marker of apoptosis in cells and is typically used in combination with a marker of cell death, in our case PI. This allows healthy, apoptotic, and necrotic cells to be distinguished from one another.
- CFSE Staining of Leukocytes
- CFSE can be used to track cell division, monitor phagocytosis, and for a multitude of other applications.
- CFSE can be used to track cell division, monitor phagocytosis, and for a multitude of other applications.
- CFSE Staining of E. Coli
- CFSE-stained bacterial cells are an extremely affordable and effective macrophage target. They can be prepared from any leftover bacteria, and heat-killing them enhances phagocytosis even more.
- CFSE-stained bacterial cells are an extremely affordable and effective macrophage target. They can be prepared from any leftover bacteria, and heat-killing them enhances phagocytosis even more.
- Intracellular Cytokine Staining
- This protocol is a modification of standard cell staining techniques that allows the detection of intracellular cytokines.
- This protocol is a modification of standard cell staining techniques that allows the detection of intracellular cytokines.
- Phosphoflow
- Phosphoflow is another intracellular staining method that allows the detection of intracellular phosphoproteins. Phosphoflow can be a valuable tool to examine cell signalling pathways.
Molecular Biology Protocols
- CALR Del and CALR Ins Screening
- This protocol detects calreticulin type I (del52) and type II (ins5) mutations, as well as many of the less common deletion mutations (e.g. del61, which is found in the Marimo cell line). We primarily use this protocol to identify mutations in patient samples, but it may be adapted to screen colonies from CFC assays as well.
- This protocol detects calreticulin type I (del52) and type II (ins5) mutations, as well as many of the less common deletion mutations (e.g. del61, which is found in the Marimo cell line). We primarily use this protocol to identify mutations in patient samples, but it may be adapted to screen colonies from CFC assays as well.
- Colony PCR - Jak2(V617F) Screening
- This protocol describes high-throughput JAK2 screening of the resultant colonies from CFC assays.
- This protocol describes high-throughput JAK2 screening of the resultant colonies from CFC assays.
- Isolation of Genomic DNA from Peripheral Blood
- This is a quick and dirty DNA extraction protocol that we primarily use for routine genotyping of our transgenic colony, but it can be used for other applications when high-quality DNA is not necessary.
- This is a quick and dirty DNA extraction protocol that we primarily use for routine genotyping of our transgenic colony, but it can be used for other applications when high-quality DNA is not necessary.
- Jak2V617F Knock-In Genotyping
- Use this protocol for routine genotyping of the Jak2V617F knock-in colony. This protocol is optimized for DNA samples isolated using our genomic DNA extraction from blood protocol (see above).
- Use this protocol for routine genotyping of the Jak2V617F knock-in colony. This protocol is optimized for DNA samples isolated using our genomic DNA extraction from blood protocol (see above).
- qPCR - Analysis of Gene Expression Using the Pfaffl Method
- This protocol is supplemented by our RNA Extraction and cDNA Synthesis protocol. We use the Pfaffl method (also known as the efficiency method) as it is more precise than the ΔΔCT method, but requires less work than the standard curve method.
- This protocol is supplemented by our RNA Extraction and cDNA Synthesis protocol. We use the Pfaffl method (also known as the efficiency method) as it is more precise than the ΔΔCT method, but requires less work than the standard curve method.
- RNA Extraction and cDNA Synthesis
- This is a general protocol describing the extraction of RNA by Trizol, followed by cDNA synthesis. We typically use this protocol to isolate RNA from bone marrow, MNCs, or cell lines, but it can be adapted for other applications.
- This is a general protocol describing the extraction of RNA by Trizol, followed by cDNA synthesis. We typically use this protocol to isolate RNA from bone marrow, MNCs, or cell lines, but it can be adapted for other applications.
- Site-Directed Mutagenesis
- We use the QuikChange II XL kit from Agilent, with some minor modifications, for site-directed mutagenesis of our plasmids.
- We use the QuikChange II XL kit from Agilent, with some minor modifications, for site-directed mutagenesis of our plasmids.
- Western Blotting
- From preparing whole cell lysates to imaging your membrane, this is your one-stop protocol for all things immunoblotting.