Step 1: Collect 2 ml of blood
Gently collect a small sample of blood, ensuring proper aseptic technique.
Step 2: Prepare density gradient medium
Carefully layer a density gradient medium, such as Ficoll or Histopaque, in a centrifuge tube.
Step 3: Add blood sample
Gently add the 2 ml blood sample on top of the density gradient medium, being cautious to avoid mixing the layers.
Step 4: Centrifuge
Centrifuge the tube at an appropriate speed and duration to facilitate the separation of blood components based on their densities.
Step 5: Collect PBMC layer
After centrifugation, you will observe distinct layers in the tube. Carefully collect the layer containing peripheral blood mononuclear cells (PBMCs) using a pipette or transfer pipette. This layer is typically found between the plasma and the density gradient medium.
Step 6: Wash and resuspend PBMCs
Transfer the collected PBMCs to a new tube and wash them with a suitable buffer solution. Centrifuge the tube again to pellet the cells, then resuspend them in a desired medium or buffer for further experiments.
Step 7: Flow cytometry analysis
To visualize the PBMCs and explore their characteristics, run the sample on a flow cytometer. Observe the unstained PBMCs, capturing their natural fluorescence and cell surface markers. Gate the lymphocyte population using appropriate markers and parameters.
Step 8: Analysis and interpretation
Analyze the flow cytometry data to gain insights into the composition and characteristics of the isolated PBMCs. Interpret the results to uncover valuable information about the immune cell populations within the sample.
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