Reducing Platelet Contamination
Platelets (thrombocytes) are small, irregularly shaped clear cell fragments derived from megakaryocytes and are necessary for normal blood clotting. Platelets by nature are very sticky, and when activated, will adhere easily to other sticky cell types (i.e. monocytes, eosinophils, etc.), creating cell clumps.
Recommendations for Sample Collection
Samples containing platelets should therefore be collected and handled gently to prevent platelet activation. The following recommendations1 may help:
- When drawing blood, use a wide bore needle (e.g. 19G), and do not apply vigorous traction if using a syringe plunger.
- Use more anti-coagulant when drawing blood (e.g. collect the blood in ACD tubes).
- Do not vortex the blood sample.
- Use buffer without Ca++ and Mg++ to prevent platelet activation.
Preparation of Mononuclear Cell Suspensions
The following recommendations may help reduce platelet contamination in mononuclear cell suspensions prepared from fresh whole blood:
- When isolating the mononuclear cells during density gradient centrifugation (e.g. Lymphoprep™ or Ficoll™), avoid taking any of the platelet-enriched plasma layer.
- To wash the isolated mononuclear cells:
- Perform a slow spin on the isolated cells (120 x g, 10 min, brake off, room temperature).
- Carefully remove the platelet-rich supernatant and discard.
- Resuspend the cell pellet in fresh buffer (using a fresh pipette).
- Repeat at least twice for a total of 3 or more washes.
How to Reduce Cell Clumping in Single Cell Suspensions
This protocol describes how to reduce cell clumping in single cell suspensions by treating your sample with DNase I.
- Lim, K. G. and Weller, P. F. 2001. Isolation of Human Eosinophils. Current Protocols in Immunology. 7.31.1-7.31.7.