Immunology & Immunological Disorders Poster Session
Materials & Methods
Discussion & Conclusion
Cytotoxicity of EDTA Used in Biological Samples: Effect on Some Breast-milk Studies
Contact Person: Michael O. Ogundele (firstname.lastname@example.org)
Materials and Methods
Reagents and buffers
Gelatin-veronal-buffered saline containing 0.15mM of Ca++ and 1mM of Mg++ (GVB++), as well as GVB2-- were prepared as described (12). In addition, sucrose-gelatin-veronal-buffered saline (SGVB++) containing 50mM of Ca (SGVB++Ca50) was prepared for the human breast-milk (HBM) complement assay.
Breast-milk samples were collected from lactating mothers at various postpartum (PP) periods, by manual expression or a breast pump. The samples were placed in sterile plastic tubes and transported to the laboratory on ice and processed immediately or stored at -70°C until use. One set of milk samples was collected in EDTA solution, to a final concentration of 20mM, while the other set of samples from the same mother was collected in an equivalent volume of PBS buffer only. 1-ml aliquots of whole-milk with and without EDTA were kept at -70°C until use. The samples were classified according to postpartum period of lactation as follows (11); colostrum (1-4 days PP, 5 samples), transitional milk (5-30 days PP, 4 samples) and mature milk (>30 days PP, 4 samples).
De-fatted milk samples were obtained by dividing whole milk samples into smaller portions and centrifuging them consecutively at rates of 500g and 3,000g at 4°C for 15 min, aspirating the aqueous phase of the milk each time. During centrifugation, the milk separates into three layers, i.e. a cell pellet, a middle aqueous layer and an upper fat layer. The aqueous layers were collected each time and processed immediately or stored as small aliquots at -70 or -80°C.
Certain portions of the de-fatted breast-milk were also further clarified by centrifugation twice at 20,000g for 15 minutes each at 4°C. The clarified phases were collected and stored in 1 ml aliquots at -70°C until required.
Human milk cell count
Total cell counts of breast milk samples were carried out under the light microscope. 1-2 ml of milk was centrifuged at 250g for 15 min. Cell pellets were suspended in 25ml isotonic PBS and washed 3 times, and then resuspended in Eagle´s minimum essential medium supplemented with 10% fetal calf serum (MEM/FCS) at 2.5x103 leucocytes/ml. The cell pellets were washed and resuspended in PBS after centrifugation at 250g for 20 min. The total white cells were counted but no differential count was done. Viability was confirmed using trypan blue dye exclusion.
Sensitized sheep red blood cells
Sheep red blood cells (SRBC) (ORAW 30/31) obtained from Behringwerke, Marburg, Germany were washed twice in GVB2+ buffer and resuspended in the same buffer. The SRBC concentration was standardized sphectrometrically by adding 100µl of the SRBC suspension to 1.9ml water and reading the absorbance at 541nm (A541)with a spectrophotometer (Stasar III Gilford instrument laboratories, Inc. Oberlin, Ohio) GVB++ was added to the SRBC until the A541 was 0.468, corresponding to an approximate cell suspension of 1 x 109/ml.
A sensitized cell suspension was prepared by a dropwise addition of an equal volume of anti-Forsman hemolysin) diluted 1:300 in GVB++ containg 20mM EDTA. The cells were incubated at 37°C for 30 minutes and at 0-2°C for another 30minutes. The cells were then washed once in SGVB++ and resuspended in this buffer. A final suspension of 5 x 108 cells/ml, obtained by an equal addition of an equal volume of buffer, was employed in the test assays.
Human breast-milk modified haemolytic assay
A micro-modified CH50 assay (12) was developed and used for the assessment of the human breast-milk Complement (C) system activity. Briefly, the sensitivity of the test assay was increased twenty-fold by reducing the total volume of the standard reaction mixture by the same factor, from 7.5ml to 375µl, while retaining the relative proportion of the buffer, sensitized sheep red blood cells, and the test samples. Previously diluted or undiluted whole or de-fatted milk was pipetted into the assay tubes with 40µl of 5 x 108 sensitized SRBC/ml and incubated for 2 hours, with occasional mixing, in a water bath at 37°C. Sucrose-gelatin-veronal buffer were used for the breast-milk assays, to further enhance its sensitivity. The reaction was stopped by addition of 1 ml of cold Gelatin-Veronal buffer (GVB2--), with or without 10mM EDTA. The cells were pelleted after the reaction by centrifugation at 12,000g for 5 min and 200µl of each supernatant were pipetted into a 96-well flat-bottomed microtitre plate (Greiner, Frickenhausen, Germany) and the amount of the released haemoglobin recorded by a plate spectrophotometer (MR 700 microplate reader, Dynatech, Torrance, CA, USA) at a test wavelength of 410nm against a reference wavelength of 630nm. From the values of optical densities (OD) obtained, the degree of haemolysis produced by a given volume of HBM was obtained from the equation:
Y = Test OD - spontaneous lysis OD - HBM blank OD X 100
H2O lysis OD - spontaneous lysis OD
Human milk total protein concentration
The total protein content of human breast-milk samples was estimated using the Bradford method (13). Precinorm U (PNU) obtained from Boehringer (Mannheim, Germany) was used as a standard. 20µl each of serially diluted standard and milk samples were pipetted into flat-bottom 96-well mirotitration plates (Greiner, Frickenhausen, Germany). 80µl of Bio Rad Protein assay colouring reagent solution was then added to each well. The plate was read after thorough mixing for 5-30 min at l = 630nm against a reference of l = 490nm in the. The values of total protein concentration were determined using a software computer programme developed for the assay.
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