Peripheral Blood Mononuclear Cells - PBMCs

What are PBMCs- Peripheral Blood Mononuclear Cells

Peripheral Blood Mononuclear Cells (PBMCs) are all blood cells with a single nucleus, which include lymphocytes (T cells, B cells, and NK cells) and monocytes. Erythrocytes, platelets and granulocytes do not belong to the PBMCs because they have either no or multiple nuclei. PBMCs are crucial in the immune system and pose important objects and tools for immunological research.

PBMC- peripheral blood mononuclear cells

Antigen Presenting Cells - APCs

Antigen Presenting Cells (APCs) are immune cells that process and present antigens to T-cells, initiating an immune response. Major types of professional APCs include dendritic cells, macrophages, and B cells. These cells display antigens bound by MHC (major histocompatibility complex) on their surface. Helper T cells and cytotoxic T cells recognize the presented antigens by specific T cell receptors. Antigen presentation is a crucial part of the adaptive immune response.

Types of APCs - professional antigen presenting cells

Antigen-Specific T Cells

Antigen-specific T cells are T lymphocytes that recognize and bind a specific antigen presented by APCs. T cells play a pivotal role in the adaptive immune response, aiding in the targeting and elimination of pathogens.

mechanism of t cell activation and differentiation

PBMC Isolation Protocol*

The isolation of PBMCs is typically performed using density gradient centrifugation. Here's a general PBMC isolation protocol:

  1.  Collect Blood Sample: Obtain venous blood and add an anticoagulant such as EDTA or heparin.
  2. Dilute Blood: Dilute the blood sample with an equal volume of PBS (Phosphate Buffered Saline).
  3. Layer Over Density Gradient: Carefully layer the diluted blood over a density gradient medium, such as Ficoll-Paque.
  4. Centrifuge: Centrifuge the tubes at 400-500 x g for 30-40 minutes at room temperature without brake. 
  5. Collect PBMC Layer: After centrifugation, collect the PBMC layer at the interface between the plasma and density gradient medium. 
  6. Wash Cells: Wash the collected PBMCs with PBS or culture medium by centrifuging at 300 x g for 10 minutes. Repeat the wash step to ensure removal of any residual density gradient medium.

PBMC isolation via density gradient

Freeze-Thawing and Cryopreservation of PBMCs*

Cryopreservation Protocol

  1.  Preparation: Prepare PBMCs at a desired concentration in a freezing medium (e.g. 10% DMSO, 90% FBS).
  2. Aliquot: Aliquot the cell suspension into cryovials.
  3. Controlled-Rate Freezing: Freeze cells at a controlled rate of -1°C per minute using a programmable freezer or a freezing container placed in a -80°C freezer.
  4. Storage: Transfer the vials to liquid nitrogen storage for long-term preservation.

Thawing Protocol

  1. Warm Up Vial: Quickly thaw the cryovial in a 37°C water bath until a small ice crystal remains. 
  2. Dilute: Immediately dilute the cell suspension with pre-warmed culture medium. 
  3. Wash: Centrifuge and wash cells to remove DMSO. 
  4.  Resuspend: Resuspend cells in culture medium and incubate at 37°C with 5% CO2. 

Activation and Expansion Protocols*

BMC Activation & Expansion

Here's a general protocol: 

  1. Culture Medium: Suspend PBMCs in a suitable culture medium such as RPMI-1640 supplemented with 10% FBS.
  2. Add Antigen/Stimulus: Add the specific antigen or mitogen (such as PHA, ConA, or anti-CD3/CD28 antibodies) to the culture.
  3. Incubate: Incubate the cells at 37°C with 5% CO2.
  4. Monitor: Monitor cell proliferation and activation markers using flow cytometry. 

Antigen-Specific T-Cell Activation & Expansion

Here's a general protocol:

  1. Initial Activation: Co-culture PBMCs with APCs loaded with the specific antigen.
  2. Expansion: After initial activation, expand T-cells in the presence of IL-2 or other growth factors.
  3. Restimulation: Restimulate T-cells periodically with peptide-loaded APCs to maintain and expand antigen-specific T-cell populations.

APC Loading Protocol*

  1. Prepare APCs: Isolate APCs (e.g., dendritic cells) from PBMCs or other sources. 
  2. Load with Antigen: Incubate APCs with the desired antigen or peptide at 37°C for 1-2 hours. 
  3. Wash: Wash the APCs to remove excess antigen. 
  4. Co-Culture: Co-culture the antigen-loaded APCs with T-cells to initiate specific T-cell responses. 
* All protocols on this page are general outlines of the procedure. Please test your protocol and adjust to your experiment and specifications. 

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