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! 

Thursday, October 24, 2013

Lowry Assay Principle and procedure

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P)

Why Lowry? 

Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes).
Other assays like Bicinchoninic Acid and Dye binding assays are used in only specific circumstances and mostly not preferred due to its sensitiveness to contaminants like carbohydrates, lipids, etc.,

 Principle behind Lowry's Assay for protein with procedure

The reactions that occur in Lowry assay are binding of Copper to the Nitrogen in the peptide. And, the phosphomolybdic tungstic acid in the Folin Ciocalteau reagent gets reduced to hetero poly-molybdenum blue by the copper catalyzed oxidation of aromatic amino acids in the peptide, in alkaline conditions. The assay must be done at the pH of 10 to 10.5 as it is sensitive to pH changes.

Reagents Required:

A) 2% of sodium Carbonate (50 ml) + 0.1 N NaOH solution (50 ml)
B) 10 ml of 1.56 CuSo4 + 10 ml of 2.37% Sodium potassium tartarate

Lowry's Reagent = 2 ml of (B) + 100 ml of (A)
Folin's Reagent what we use in our laboratory is ready made one (2 N) which is just diluted and made as 1 N for use. (by mixing with equal volume of water)

For constructing Standard curve, BSA is generally used. Stock of 1 mg/ml is required. 

Note: Prepare all the reagents in distilled water. And, prepare the reagents freshly before use, just before use, to avoid precipitation of the salt added, also mix the reagents A and B only before use. 

  • First the BSA stock is diluted for standard curve construction. Let the total volume be 1 ml, so for preparing a concentration of 0.05 ml, take 0.05 ml of 1 mg/ml BSA stock and mix with 0.95 ml of distilled water.
  • Similarly, we can prepare various standard concentrations like 0.1,0.2,0.4,0.6,0.8,1 mg/ml for  total volume of 1 ml by mixing 0.1, 0.2,0.4,0.6,0.8,1 ml of stock BSA with 0.9,0.8,0.6,0.4,0.2,0 ml of distilled water respectively. 
  • From this prepared standard concentrations of BSA, 0.2 ml must be taken for assay. For example, from the 1 ml of 0.05 mg/ml BSA prepared 0.2 ml must be taken in a separate test tube. Similarly, 0.2 ml must be taken from all the other standards.
  • Then, 2 ml of Lowry's reagent must be added to each of this 0.2 ml sample and incubated for 10 minutes.
  • Then, 0.2 ml of Folin's reagent is added to each of the incubated tubes and incubated for 30 minutes.
  • After 30 minutes, the tubes will have blue colored solution, which is further read at  660 nm in Spectrophotometer. 
  • Graph should be plotted with absorbance in y axis and concentration in X axis. 
And, using this standard graph, we can determine the concentration of unknown sample by extrapolation.

This is a very easy assay which I learnt in the very beginning  of my course, but, still a very useful one and I'll be using it even after years I believe. 

You know, I feel the major disadvantage of this Lowry assay is that you need to spend at least two hours to complete it including reagent preparation and incubation. (Sometimes, I feel like, oh, no, incubation for 30 minutes!How good it would be if there is no need for incubation?) And, when you don't have that much time, what will you do for assaying your protein? 

It's simple, just measure absorbance at 260 nm and also 280 nm. Using the formula,

Protein (mg/ml) = 1.55 *(Absorbance at 280 nm) - 0.76 * (Absorbance at 260 nm).

This can be used when you have a spec which operates in UV range. No need for doing Lowry Assay, Yippee :P 

Hope, this helped you :) Found any mistakes? Doubts? Let me know with your comments!

Wednesday, October 2, 2013

Cre lox system - Basics

It's been a long time, since I wrote a post here. Finally, final year of my course and so very busy :) with books! And now, got time to share with you.

As you know already (don't know?, then get to know :P ), I use to have favorite subjects in each semester, third semester it was microbiology, then in fourth semester, it was cell biology, fifth was molecular biology, sixth was genetic engineering and now i'm in seventh semester, but, you know what, I don't have a favorite subject!!! Don't worry that I lost interest, instead, I got 3 favorite subjects - Immunology, Animal biotechnology and Plant Biotechnology!

It's awesome this time to have more than one favorite subject and here I'm gonna tell you something about, cre-lox system which is most widely used for making modifications (mostly deletions). These modifications can be done at a specific tissue alone by using a tissue specific promoter i.e., you can selectively knock out a particular gene in particular cells (for eg: hepatocytes) alone.

Cre recombinase is a protein which could bind with specific sequences called loxP sequences. To explain it in a simple way, the cre recombinase binds at loxP sites on either side of your gene (which is to be deleted) and makes a cut! This leads to removal of the gene and further the break is repaired using the DNA ligase of host system.

A single loxP site will have 13bp on either side of middle 8bp. The cre recombinase protein binds with this 13bp sequences forming dimer i.e., one loxP site contributes for a dimer (containing 2 cre recombinase units). Similarly, loxP on the other side of the target gene will contribute for a dimer formation. Then these two dimers will form tetramer (by folding the DNA strand as shown in figure. Then, cutting at the tetramer site happens leading to either translocation, inversion or deletion depending on the direction of the loxP site (i.e whether it is direct or invert repeat on either side of the target gene or located on different chromosomes)

                         13bp                          8bp                  13bp

This insertion, deletion and translocation could be done specifically at a particulat type of cell by using specific promoters. For example: To delete a particular gene in kidney cells alone, one could use kidney specific promoter for controlling the production of cre recombinase, so that cre recombinase will be expressed only in kidney cells. The molecular mechanisms of this deletions and recombinations invloves holliday junction formation. But, to be frank, i'm not clear or good at this holliday junction.

I''m trying to understand the molecular mechanism behind this, and, once I'm clear with it, I'll share it with you.

Understood the cre-loxp system? Got  any doubt? anything wrong in my explanation? Kindly comment and I would try to reply or correct as soon as possible.