Monday, October 28, 2013

Isolation of monocytes from PBMC (Peripheral Blood Mononuclear Cells) - Principle and protocol

Whenever I'm made to realise that I'm not clear enough or good at something, I try to make myself clear with it. It happened today, during my laboratory examination, I was asked to perform monocyte isolation from a given blood sample, but, unfortunately, I was not very clear with the principle behind it.(but, still I managed to complete the experiment as I know the protocol, but, knowing the principle behind each step of the protocol clearly is very important, isn't it?). But, nothing is wrong in it, I made myself clear with it now. That's good, right?

So, let me share with you some basic principle and protocol for isolating monocyte from blood sample.

For isolating monocytes, initially we must isolate PBMC (Peripheral bood mono nuclear cells) from the blood sample. Here, let us make few terminologies clear before starting with the principle.

Peripheral blood sample - It is the blood sample obtained from acral areas of body (in general, it is the blood collected from hands by your doctor for doing blood test).

Peripheral blood mononuclear cells (PBMCs) - It is the cells which have a prominent round nucleus which is present in the peripheral blood sample. Note, all the blood cells have a single nucleus, i.e, mono nucleus, but, neutrophil, basophil and eosinophils have lobed nucleus, i.e. they are polymorphic. Lymphocytes and monocytes have round nucleus and they are referred as PBMCs.

Ficoll hypaque - It is a polysaccharide which is soluble in aqueous solutions and widely used for performing separations based on density. Here, we can isolate PBMC's from RBC's and other blood cells based on the difference in their density.

Percoll Gradient - Percoll is silica particles coated with PVP (polyvinyl pyrollidine) and preferred for biological isolations because of its characteristics like non toxicity and low osmolarity. Besides it's use in monocyte isolation it is also used in sperm selection in assisted reproduction where healthy and active sperm cells can be selected using Percoll gradient.

Isolation of PBMCs

Protocol in short - (if 1ml of blood sample is taken, dilute with 1ml of 1X PBS before starting, when you use 10XPBS it is reported that the separation is not efficient, problem occurs while overlaying ficoll hypaque)

Take 2ml of ficoll hypaque for isolating PBMCs from 4ml of diluted blood (2ml blood diluted with 2ml of 1X PBS) in a falcon (always maintain 1:2 ratio of Ficoll and blood sample). Overlay the Ficoll hypaque with diluted 4ml of blood sample using pipette (do it slowly to avoid mixing of the blood with ficoll due to force given during addition). Centrifuge this at 1400rpm for 30 minutes at 20 degree C (brake off) 

After centrifugation, you can find buffy coat containing PBMCs over the Ficoll layer, as shown in the figure below: (hisep is commercial name of Ficoll)

Carefully the plasma layer must be discarded and the buffy coat is collected in a separate clean falcon (must be done with care to avoid mixing of layers). The separation of the cells is based on their density, Platelets are the least dense cells and they top the layers, followed by PBMCs which are also not dense enough to cross the Ficoll whereas RBCs and other WBCs are dense enough to cross the Ficoll. 

The separated buffy coat is further washed with suitable medium at 1400rpm, 15minutes, 20 degree C (brake on). Again resuspend the pellet in appropriate medium and do Trypan Blue viability check assay and count the viable cells and  find the number of viable cells.

And, normally the yield will be 0.5to 3 *10^6 cells/ ml.

Isolation of monocytes using Percoll gradient

Prepare Percoll gradient (using Standard Isotonic Percoll 9parts Percoll + 1 part 10X PBS) by using 0.15M NaCl. Generally, 60% (lower layer), 45% (middle layer) and 35% (top layer) percoll solutions are prepared for preparing the gradient. For a concentration of 3*10^7 cells lower and top layer of 2.5ml and middle layer of 5ml can be used. This is prepared by layering successive layers in a falcon, if required, centrifuged. 

Then overlay the gradient with PBMC and after centrifugation at 1400 rpm for 30 minutes, monocytes can be obtained from the middle layer.

Hope, this helped you. any mistakes? Doubts? kindly comment! 

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