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Modified Method for Isolation of Peripheral Blood Neutrophils from Bovines and Humans

Raheela Akhtar 1, Zafar Iqbal Chaudhary 2 and Yongqun Oliver He 1. 1Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology,

University of Michigan Medical School, Ann Arbor, MI 48109 2 Bhaud Din Zikria University Multan, Pakistan 54000.

Corresponding author: Raheela Akhtar, teetu_meetu41@yahoo.com, raheela@med.umich.edu, Tel No: 001-734-615-2455 Fax No: 001-734-936-3235 Accepted: 12 May 2010

Abstract A modified method based on the principle of density gradient centrifugation, using lymphocyte separating media was developed for neutrophils isolation from peripheral blood of bovines and humans. The advent of this method with 95% purity, 96% viability, retained morphology and function of neutrophils showed excellent strategy for cell separation. Keywords: Isolation, Neutrophils, protocol. Introduction Microphagocytes or granulocytes of which most numerous are the neutrophils were discovered by Elie Metchnikoff when he inserted rose thorns into starfish and found that wandering mesodermal cells accumulated at the puncture site 1. These cells with phgocytic properties are the first line of defense against infection. They squeeze through the capillary walls and into infected tissue where they kill the invaders (i.e., bacteria, fungi) and then engulf the remnants by phagocytosis. This process continues even in healthy people 13. Studies have been undertaken to describe that although the bovines and human neutrophils utilize similar antimicrobial strategies a comparison of both types of cells has demonstrated distinct differences that may reflect variations in regulatory mechanisms 6, 14. Some previous studies 5, 14 cast a perspective that bovine neutrophils apparently lack an N-formyl-peptide chemotactic receptor, contain large cytoplasmic granules and antibacterial peptides. Because of these differences, the biological responses and isolation procedures of bovine neutrophils may not necessarily be identical as those of human neutrophils. Currently available methods for isolation of neutrophils are based on two principles:

1- Difference of cell specific gravity using density gradient material 4. 2- Using a neutrophils specific migratory stimulus 7.

Using density gradient material like ficoll-hypaque does not yield the pure neutrophils population as differences in buoyancy of different leukocytes are not sufficient for easy separation. Haematological variation between species makes this technique limted 12. A common method by which viable, quiescent and purified neutrophils can be isolated from different species has not been yet developed. Although, there are many methods available but none is satisfactory in terms of purity, viability, and provision of quiescent neutrophils. Though the negative immunomagnetic separation 8, yields a highly pure population of cells but it is not economical. There is need for the antibodies against specific markers, secondary antibody coated magnetic beads and column with a magnetic field which makes its application purely theoretical in field. As the beauty of sciences is in making the things simple so in the current studies we introduced a modified simple, comparatively convenient, economical method for neutrophils isolation from bovines and human blood. Materials and Methods Blood Sampling Five ml of blood samples were drawn from jugular vein of fifty healthy cows using 10ml syringe by aseptic measures and the same amount of blood was collected from fifty healthy human donors from antecubital area of the arm with sterile 10 ml needle. The blood sample was poured slowly in EDTA coated vacutainers for further use.

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Reagents and buffer Hanks Balanced Salt Solution, Dextran-500, LSM Cat # 50494 (LSM is a sterile filtered solution which contains 6.2g Ficoll and 9.4 g sodium diatrizoate per 100 ml. The density is 1.07700.1-1.0800 g/ml at 20oC). ACK lysis buffer, Trypan blue exclusion (Sigma Aldrich). All the solutions were purchased from MP Biomedical unless otherwise mentioned. Cell Separation Two ml LSM was pipette into a 15ml falcon tube and the then double volume (4ml) of anticoagulant added blood was carefully poured on this LSM layer down the side of each tube. Without shaking, the tubes were transferred to the centrifuge machine with swinging rotar and were spin at speed of 652xg for 10 minutes at 4°C. Four different layers of blood were formed. The sediment containing neutrophils and erythrocytes was taken by discarding the supernatant layer containing lymphocytes, monocytes and plasma layer on top respectively. The sediment was mixed with equal volume of 6% dextran (in Normal Saline) and was incubated at 37°C for 45 minutes to allow the sedimentation of erythrocytes. The pellet containing erythrocytes was discarded and the neutrophils rich supernatant was collected and was mixed with an equal volume of HBSS. The reaction mixture was centrifuged at 290xg for 10 minutes at 4°C. The pellet containing the neutrophils was taken by removing the supernatant. Washing with HBSS was repeated once or twice depending on the amount of erythrocytes present as a contaminant. 1ml ACK lysing buffer was added in the pellet to lyse the remaining R.B.Cs. The tubes were incubated at 37°C for five minutes. Double volume of HBSS was added and the tubes were spin at 652xg for 10 minutes at 4°C to remove the lysed particles of erythrocytes. Again the supernatant was removed by taking the pellet. Quantifying cell number and viability The final neutrophils pellet was resuspended into desired volume of PBS to achieve the appropriate cell concentration usually 2 ml. 10µl of this cell suspension was diluted with 190ul of 2% acetic acid. Few microliters of this mixture were dropped on hemacytometer and the neutrophils were counted in the 25 squares inside the central double lines. The neutrophils count was divided by 25 to obtain the average per square. The average was multiplied by 5x106 and then by the volume of final cell suspension (2ml) to determine the total number of isolated neutrophils. Cell viability was determined by mixing equal volumes of neutrophils suspension and trypan blue, pipetting the mixture onto microscopic slides and viewing the cells under microscope. Cells that exclude the trypan blue and appeared transparent were counted as viable, whereas cells that turned blue were counted as dead. Analysis of purity. Purity was evaluated with hemocytometer by differential counting of neutrophils vs nonneutrophils and performing flow cytometery using anti bovine CH138A and CD15 antibodies.

Results

The neutrophils were identified by their multilobular nuclei on hemacytometer and by identification of surface markers specific antibodies. The purity of neutrophils for both species calculated was 95% and viability was upto 96% (Fig 1a and 1b).

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CH138A negative cells CH138A positive neutrophils Fig 1b: The histogram showing the separation of CH138A positive neutrophils and IgG negative control.

The neutrophils isolated by this method maintained their morphology (Fig 2a and 2b) and functionality (data not shown).

Figure 2a: The human neutrophils separated by modified method

Figure 2b: The bovine neutrophils separated by modified method Discussion The bulk of information derived from the previous studies show that the cell isolation methods including the sedimentation of whole blood through dextran, centrifugation of leukocyte rich plasma and lysis of erythrocytes in final neutrophils prepration are imprecise methods as they alter the neutrophils function. The study presented a simple alternative and comparatively rapid method for isolation of different types of neutrophils from humans and bovine peripheral blood with least erythrocytes contamination. Most methods use not less than 20ml. The development of newer and novel techniques for isolation of bovine and human neutrophils is important to study the characteristics of these cells and their use in applied immunology. Previously a convenient and rapid separation using centrifugation through a Ficoll-sodium metrizoate solution by layering the diluted blood over the ficoll-sodium metrizoate solution and centrifugation at a low speed for a short time was introduced 3. But it could not be successful due to density related issues of ficoll. Modifications of thethis

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formulation have been made by numerous workers. "LSM" produced by MP Biomedicals, has a unique formulation using the successful substitution of sodium diatrizoate for the sodium metrizoate. The development of newer and novel techniques for isolation of different species neutrophils is important to study the characteristics of these cells and their usage in applied immunology. This study concentrates an additionally reasonably fast, simple, alternative and comparatively economical mean for isolation of neutrophils from different species. The ready availability of reagents, usage of small amount of blood high purity and viability makes this approach a friendly and handy method to use. Moreover, this method is suitable for isolation of different types of neutrophils like humans and bovine neutrophils with same sensitivity. As the heparin is considered to produce poor results than EDTA therefore EDTA was preffered for this study. 11 It is less laborious and one just needs a centrifuge machine with few chemicals to perform it. As in more time taking and vigorous methods the chances for extremely sensitive neutrophils isolation decreases because of their short life span and delicacy, this method helps to minimize that risk. This study revealed the simplicity of the technique used as compared to the previous studies 10 new cell isolation techniques based on the use of monoclonal antibodies against specific target cell epitopes followed by cell sorting or magnetic separation. This biomagnetic bead isolation of cells is a rapid method that allows for the separation of many specific cell types but depending on availability of proper monoclonal antibodies that are not economical and easily available 9. Moreover, the utility of this technique for isolation of bovine cells has not been evaluated. Self prepration of ficoll-hypaque provides lesser numer of cells if the solution is not of appropriate specific gravity it may hamper the isolation of cells. In addition to it according to studies of 15 improper densities of ficoll also change the morphology of cells and has high erythrocytes contamination. Therefore, keeping the above discussed points in mind our method may be of great utility to most labs studying the biochemistry of these cells. We concluded that this modified method is a midway solution for the problems encountered by currently used methods as the density of widely used ficoll-hypaque is not fixed and varies with extraneous factors and isomagnetics isolation is more expensive and complicated. Therefore, this new method can be a better future option for many neutrophilic studies in terms of simplicity, high purity, and repeatability, irrespective of the anticoagulant used. Acknowledgements We are thankful to Professor Abdul Ghaffar, University of South Carolina for his guidance and technical help. References

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