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Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification
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Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Negative Selection Preparation of CD4 Positive T Cells

Introduction to Magnetic Separation Techniques

  • Melanie Matthew introduces the Max Mini magnet and its application in cell separation, specifically for CD4 positive T cells using military biotechnology kits.
  • The Midi Max is designed for larger separations, capable of processing up to 10^9 cells, while the Mini Max is suited for smaller volumes.

Cell Preparation Process

  • In negative selection, the target cells pass through a column while unwanted cells are retained; the elutant contains the desired cells.
  • Emphasis on maintaining sterility during procedures if cells are intended for culture; techniques shown can be adapted for various cell types.

Lymph Node Processing

  • Lymph nodes from a mouse are ground into a single-cell suspension using a 70-micron cell strainer to ensure no clumps remain.
  • After grinding, cloudy media indicates successful extraction of lymphocytes; washing with media helps maximize yield by removing stuck cells.

Cell Pelleting and Red Blood Cell Lysis

  • The pelleted single-cell suspension is prepared by centrifugation at 300g for 10 minutes; consideration of dead cell populations may require additional removal steps.
  • RBC lysis involves adjusting osmolarity carefully to avoid lysing lymphocytes; quick addition of solutions is crucial.

Final Steps in Negative Selection

  • Post-RBC lysis, counting lymphocytes on a hemocytometer determines antibody requirements for negative selection.
  • Initial incubation with biotin and antibody cocktail precedes adding antibiotin microbeads before proceeding with magnetic separation using an LS column.

Cell Separation Process Using Magnetic Beads

Preparation and Initial Steps

  • The process begins with the use of a column where liquid is allowed to drip out. It's noted that surface tension will prevent the column from running dry until cells are added.
  • After a 15-minute incubation with antibiotin magnetic beads, the cells and Max buffer are spun down. The buffer is then poured off, and cells are resuspended in an appropriate volume based on cell count as per the melatonin protocol.

Cell Application and Collection

  • The cell suspension is applied to the rinsed column. This setup is for negative selection, meaning that CD4 positive T cells will flow through while other cells are retained.
  • Following application, the column is washed with 12 mL of Max buffer to collect only CD4 positive T cells in the elution.

Verification of Purity

  • After spinning down the collected CD4 positive T cells, they are resuspended and labeled with a dye suitable for two-photon imaging.
  • A small sample of purified cells is also labeled with anti-CD3 and anti-CD4 antibodies for flow cytometry (FACS), ensuring a clean population of CD4 positive T cells has been obtained.
Video description

Reference: https://app.jove.com/v/409/isolation-cd4-t-cells-from-mouse-lymph-nodes-using-miltenyi-macs The process of isolating CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification involves several steps. First, the lymph nodes are harvested from the mouse and processed to obtain a single-cell suspension. The suspension is then incubated with CD4-specific antibodies conjugated to magnetic beads. These beads bind specifically to the CD4+ T cells. The suspension is then passed through a magnetic column, which retains the CD4+ T cells while allowing other cells to flow through. The retained CD4+ T cells are then eluted from the column, resulting in a highly purified population of CD4+ T cells ready for further analysis or experimentation. This method allows for the isolation of CD4+ T cells with high purity and yield, making it a valuable tool for immunological research. Additionally, the Miltenyi MACS purification system offers the advantage of being quick and easy to use, with minimal hands-on time and no need for expensive equipment. This makes it a convenient and efficient method for researchers working with mouse models to study immune responses, autoimmune diseases, and other immunological processes. The isolated CD4+ T cells can be used for a variety of downstream applications, such as flow cytometry, cell culture, functional assays, and gene expression analysis. Overall, the Miltenyi MACS purification system provides a reliable and effective way to obtain pure populations of CD4+ T cells for in-depth immunological studies. Furthermore, the Miltenyi MACS purification system is highly customizable, allowing researchers to tailor the purification process to their specific experimental needs. This flexibility enables researchers to isolate CD4+ T cells from various sources, including blood, lymph nodes, and spleen, with high purity and yield. The system also offers the option to isolate different subsets of CD4+ T cells based on specific surface markers, providing researchers with the ability to study distinct populations of T cells in their experiments. In addition, the Miltenyi MACS purification system is compatible with a wide range of antibodies and reagents, allowing for seamless integration into existing experimental workflows. This compatibility ensures that researchers can easily incorporate the system into their studies without the need for extensive optimization or revalidation.