Blood and Erythrocyte Fractionation

The unique structure of blood makes it very easy to separate red blood cells from plasma and the other formed elements by differential centrifugation. Once isolated, red cells can be lysed (burst open) by suspension in a hypotonic medium. They take up water by osmosis and eventually explode, leaving an empty membrane sack (sometimes called a "ghost") behind.

Before conducting a fractionation for the first time, you should review the principles behind tissue fractionation and quantitative methods related to liquid measurement and handling, protein assays, and determination of protein yields.

Materials

When tissues are collected for fractionation it is usually important to record the species and to record the source of the animal(s) if the strain of animal might make a difference. We have used caninie blood for this preparation, and domestic dogs are all one species (Canis familiaris). The structure of erythrocytes is not likely to differ from one breed of dog to another, therefore we haven't always reported a specific source or breed. However, an editor may have to check that your source is legitimate (kidnapping your neighbor's collie for a blood sample would not be legitimate), and readers will want to know where they can obtain the stuff. If you use a commercial source, report the source in the materials/methods. I might add that we do not always use the same species for this study. So far we have used whole dog, rabbit, pig, and horse blood.

The target fraction and plans for the material dictate the volume of tissue that should be used. You would need a very large volume if you were to try to purify a specific protein that is of low abundance, regardless of the intended purpose. Purification procedures result in losses of material, so the necessary volume of starting material increases with the need for purity of a suspension of organelles, or, say, a specific enzyme. For a qualitative analysis such as characterization of membrane associated proteins by SDS-PAGE, a small quantity of starting material should be sufficient.

Unless the intention is to collect serum, whole blood must be treated with anticoagulant and either used immediately or stored refrigerated until use. A suitable isotonic (iso-osmotic) buffer for preparing washed red cells consists of 0.9% NaCl with 5 mM sodium phosphate, pH 8. A suitable hypotonic buffer for lysing red cells consists of 5 mM sodium phosphate, pH 8, without NaCl. Distilled water alone would be fine, but you would have no control over pH. If the procedure is conducted at ice bucket temperature and is carried out fairly quickly, no preservative should be needed.

Red cell isolation

We have had good results by conducting the fractionation directly in centrifuge tubes that hold three or so volumes of buffer in addition to the original whole blood sample. One volume refers to the initial volume of sample. Thus if the volume of whole blood is three milliliters, three volumes of isotonic buffer would be 9 ml. For solid tissues one volume is one ml per gram tissue. Any fractionation procedure results in losses due to incomplete separation of components, materials sticking to surfaces, and fluids left behind after transfer. The smaller the final pellet, the more likely too much of it will be lost through attrition. Our centrifuge tubes hold a maximum of 15 ml. We can fit 13 ml into them without spilliage during centrifugation, so we typically start with two or three ml of whole blood and bring the volume to13 ml with isotonic buffer.

A buffer that is considered to be isotonic for a cell type has the same osmolarity as the environment in which the cell is normally found. Thus it neither swells nor shrinks (crenates) in the buffer. The major electrolyte in blood plasma as well as in the interstitial fluid in vertebrates is sodium choride, so the logical choice for an isotonic buffer is 0.9% NaCl. It is necessary to mix the sample thoroughly with buffer to begin the process of washing the red blood cells free of plasma proteins. An effective means of mixing is trituration. Trituration refers to repeatedly pulling liquid into a pipet and ejecting it, while keeping the tip immersed. The suspension should be centrifuged immediately following trituration. Spinning down the cells separates the cell pellet from most of the plasma proteins, which are soluble and remain in the supernatant. Cell pellets are seldom tightly packed, however, so without one more wash there may still be plasma proteins in with the pellet.

Centrifugation at 600 x g brings down the red cells quickly. The low speed works because the cells are heavily packed with hemoglobin. Ten minutes is more than enough time to separate red cell pellet from dilute plasma supernatant. After removing an aliquot of supernatant the remaining liquid should be removed and total volume (including aliquot volume) recorded. The aliquot will be needed later for qualitative analysis, so it should be kept on ice until it can be stored in the freezer. We don't freeze the aliquots right away because we need to determine their protein concentrations.

The pellet should be resuspended in isotonic buffer by trituration, then re-centrifuged (this second procedure is called a wash step). We don't usually bother collecting aliquots from supernatants or record their volumes following wash steps because the protein concentration is so low relative to that of the first supernatant.

Suggestions for good lab technique

• Keep up with the fractionation procedure. It is not good practice to let a preparation sit and deteriorate. If you are not working on a preparation, keep it on ice.
• When handling the preparation, hold it so that you don't warm up the centrifuge tube with your hand.
• When pellets will undergo multiple wash steps, we find it convenient to put a mark on each centrifuge tube indicating final volume, so that when we resuspend we need only fill to the mark.
• For bulk transfer of liquid we use a pasteur or transfer pipet. We use calibrated pipettes only when precise measurement of volume is necessary. It is inefficient to remove a supernatant with an automatic pipettor, and the practice causes needless wear and tear on the instrument.
• It is bad practice to dip a pipette into a stock solution of buffer, particularly when a number of people use the same stocks for their experiments. Not only is it inefficient, but one dirty pipette can ruin a stock solution for an entire lab. Unless a buffer solution is very expensive it is preferable to pour a working quantity into a smaller vessel for resuspending pellets and/or preparing samples and standards.
• To ensure that stocks remain clean, experienced personnel do not return leftover material to a stock bottle.
• The object of centrifugation is to separate components, a purpose that is defeated if they are re-mixed while pipetting the supernatant. One should not eject liquid from a pipet near the surface of a pellet. A tube removed from a fixed angle rotor should be handled gently and tilted so that the surface of the pellet is level.
• The object of a wash step is to rid individual components of contaminating solutes. If chunks remain in suspension, the wash step is ineffective. Each time, the pellet must be completely dispersed.

Lysis and recovery of membranes

If qualitative analysis (e.g., SDS-PAGE) cannot be conducted immediately, then the aliquots should be stored frozen after determining their protein concentrations.

After two "spins," the buffy layer containing white blood cells should be lost, and platelets will not have spun down as quickly as red cells, so the pellet should consist almost exclusively of red blood cells. Resuspension in hypotonic buffer lyses most of the cells. Lysis can be confirmed by watching the suspension as the pellet is triturated. Whole red cells produce a cloudy, opaque suspension. Once the cells are lysed the suspension turns clear, although it will remain a dark red. Lysis releases the red cell contents (almost entirely hemoglobin) into the buffer, leaving empty membrane sacks, or red cell "ghosts" in suspension.

The aliquot of lysed red cell suspension should be kept small, since the greater the volume of the aliquot, the less the membrane yield. Red cell membranes pellet quickly when centrifuged at 12,000 x g. The process again takes about ten minutes. The supernatant contains very concentrated hemoglobin despite having been diluted. The membrane pellet is difficult to see due to the red color of the supernatant. It must be washed several times in order to remove most of the remaining hemoglobin. After taking an aliquot of supernatant the remaining liquid again must be separated and volume determined. As lysis proceeds, ruptured cells empty their contents increasing the osmotic strength of the medium, until a balance is reached in which the medium is again isotonic with the remaining intact cells. If intact cells remain after the first resuspension in hypotonic buffer, they will lyse during the next wash.

Complication

The small "button" of material that may appear near the bottom of the tube is a tangle of fibrin, white cells, platelets, and unlysed red cells. It should not be confused with the membrane pellet, which from 3 ml whole blood should be quite large, perhaps 1-2 ml in volume (membranes do not pack as tightly as red cells). It is often difficult to see where the supernatant ends and the pellet begins, thus there is a risk of sucking the membranes up as the supernatant is discarded. If it becomes difficult to distinguish where the pellet begins, one can stop part way through the removal of supernatant, resuspend everything, and recentrifuge. With some of the dilute hemoglobin removed the supernatant should be lighter in color and the pellet more visible. It will be necessary to obtain another aliquot and take a volume measurement, since the second supernatant in this case will contain a significant amount of protein.

It will take several wash steps to clear most of the red color (hemoglobin) from the membrane pellet. The pellet is readily liquified by gentle agitation, and it can be transferred to an aliquot tube using a automatic pipettor. The membranes will stick to a glass pipette. Use of a pasteur pipette to transfer the membrane sample may result in a significant loss of material.

Protein assay

Almost any kind of analysis will require that one knows the protein concentration in each sample. It is convenient to prrepare a protein assay while conducting the fractionation, finishing up when the membrane sample is collected. We have found it convenient to use the Bradford protein assay because the assay uses up relatively little protein and because a sample can be prepared and assayed in just a few minutes.

Typical concentrations for the aliquots collected in this study are

• Plasma fraction, 4 to 10 mg/ml
• Lysate fraction, 20 to 50 mg/ml
• Cytoplasm fraction, 20 to 50 mg/ml
• Membrane fraction,1 to 8 mg/ml

Actual concentrations may fall outside of the suggested ranges, but if a measured concentration is off by an order of magnitude or so, then some mistake must have been made.

Notes on the protein assay

• One experienced individual can conduct the fractionation and protein assay in less than one hour. Two individuals who perform the procedures for the first time typically take a good three hours to finish. Dividing responsibilities can save a lot of time.
• Protein assay tubes should be kept at room temperature. Some breakdown will take place, but total protein content will be unaffected. Cold culture tubes will fog up in the spectrophotometer.
• The standard curve can be run and samples run later, but the same batch of reagent and same instrument should be used.A better practice is to add color reagents to standards and unknowns at the same time, so that they are read under the exact same conditions and in the same time frame.
• Remember to read absorbance, not transmittance.

Record keeping

Much more detail should be included in a lab notebook than one would report in the materials and methods section of a paper. For example, one reports time, temperature, and g force to describe a centrifuge run. The notebook, though, should include what machine was used and rotor type. A particular centrifuge might turn out to be out of calibration, for example, having a significant impact on your experiment. Here are examples of the kinds of records that should be kept.

• How much blood did you start with? What was the species? How was it delivered to you?
• What specific centrifuge was used and what was the rotor type?
• What specific automatic pipettor did you use (serial number is embossed on the body)?
• Did you pre-rinse the tips? How did you set volume?
• Record which spectrophotometer was used, calibration procedure, what scale was read, wavelength used.

What's next?

It is time to analyze the protein samples by SDS-PAGE. If they will be run on another day, then the samples should be stored frozen (-20C) until it is time to use them. You should run all of the fractions to assess the effectiveness of the separation. The analysis will concentrate on the membrane fraction, however.