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{"project":"2_test","denotations":[{"id":"12925238-11713907-8102422","span":{"begin":1234,"end":1236},"obj":"11713907"},{"id":"12925238-11798399-8102423","span":{"begin":1593,"end":1595},"obj":"11798399"},{"id":"12925238-7978280-8102424","span":{"begin":1736,"end":1738},"obj":"7978280"},{"id":"12925238-7978280-8102425","span":{"begin":2636,"end":2638},"obj":"7978280"},{"id":"12925238-11895795-8102426","span":{"begin":4050,"end":4051},"obj":"11895795"},{"id":"12925238-6781476-8102427","span":{"begin":4052,"end":4054},"obj":"6781476"},{"id":"12925238-14056485-8102428","span":{"begin":5644,"end":5646},"obj":"14056485"},{"id":"12925238-9231902-8102429","span":{"begin":7705,"end":7707},"obj":"9231902"},{"id":"12925238-12374734-8102430","span":{"begin":7775,"end":7777},"obj":"12374734"},{"id":"12925238-5432063-8102431","span":{"begin":7834,"end":7836},"obj":"5432063"},{"id":"12925238-388439-8102432","span":{"begin":7837,"end":7839},"obj":"388439"},{"id":"12925238-11390641-8102433","span":{"begin":7904,"end":7906},"obj":"11390641"},{"id":"12925238-7738119-8102434","span":{"begin":7993,"end":7994},"obj":"7738119"},{"id":"12925238-10651630-8102435","span":{"begin":8042,"end":8044},"obj":"10651630"}],"text":"Methods\n\nAcquisition of murine and human blood samples\nBlood from 129Sv wild type and annexin A7 knock out mice (anxA7-/-) kept under pathogen-free conditions and fed with regular food, was collected in 1 ml syringes with a 22 gauge needle punctating the right cardiac ventricle immediately after they had been killed by cervical dislocation. Coagulation generally was inhibited with 1/10 volume of 10 mM NaEDTA, pH 7.5, already present in the syringe. The mice were killed by cervical dislocation in order to avoid any effect of anaesthetic chemicals on blood parameters. The wild type and mutant mice were matched regarding their age, weight and sex. The daytime of blood collection, sampling site and collection method were kept constant during all experiments. A typical murine blood sample volume consisted of 550 μl, with variations ranging from 200 μl to 900 μl. Human blood samples from the authors were collected by vein puncture using common EDTA-monovettes (Sarstedt).\nWe performed our studies using well standardized sampling conditions, as differences in the blood sampling site, for example between tail and heart, show large changes in all cell type counts. First and second sample draw affect blood measurements, too [22]. Also the genetic background in common laboratory and transgenic mice affects the phenotype. A change from one strain to another may protect mice from effects of the primarily induced genetic defect. For example, BALB/c mice have a significantly increased velocity of platelet aggregation as compared to 129Sv mice, which were used throughout this study [23].\n\nSelf forming iodixanol density gradients of human red blood cells\nThe subcellular distribution of annexin A7 was addressed according to [24]. Red blood cells were centrifuged and washed in isotonic buffered salt solution, collected in ice cold lysis buffer (20 mM Hepes, pH 7.4, 200 μM CaCl2, 0.25 M sucrose, and proteinase inhibitors (Roche)), homogenized by a loosely fitting dounce homogenizer and additional passages through a 22 gauge needle. A 50 % iodixanol solution was established by mixing Optiprep solution (60 % iodixanol; Sigma) with buffer (120 mM Hepes, pH 7.4, 1.2 mM CaCl2, 0.25 M sucrose). Seven parts of this 50 % iodixanol solution were mixed with thirteen parts of the red blood cell solution, generating a gradient solution containing 17.5 % iodixanol. The mixture was filled into a centrifuge tube underlayed by cushions of 30 % and 35 % iodixanol and centrifuged for 3 hours at 270,000 × g in a SW41 Ti swing out rotor (Beckman) to achieve a nearly linear density gradient (approximately 1.06 g/ml to 1.20 g/ml) [24]. Fractions of 0.6 ml were collected from the top of the gradient by a 1 ml syringe without using a needle and immediately frozen in liquid nitrogen for further use.\n\nExovesicle generation from human and murine red blood cells\nRed blood cells naturally can form hemoglobin containing exovesicles. In vitro these vesicle formation is Ca2+ induced. Freshly collected lithium-heparin anticoagulated blood samples were centrifuged for 5 minutes at 400 × g and 4°C. The erythrocytes were washed five times with 20 mM Hepes, pH 7.5, and 150 mM NaCl (washing buffer). A volume of 500 μl washed red blood cells were combined with 3 ml of vesiculation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl2 adjusted to 5 μM Ca2+ ionophore A23187) and incubated for 30 minutes at 37°C. After incubation EDTA was added to the solution to a final concentration of 5 mM and centrifuged for 5 minutes at 400 × g and 4°C. The supernatant was collected and centrifuged for 20 minutes at 15,000 × g and 4°C. The resulting pellet was resuspended in washing buffer and centrifuged at 400 × g again to collect a supernatant enriched with red blood cell microvesicles. The supernatant of the 15,000 × g step was further centrifuged for 60 minutes at 100,000 × g and 4°C. The final pellet of this step was resuspended in washing buffer and centrifuged at 15,000 × g again to purify a supernatant enriched in nanovesicles according to [7,20]. The vesicles were assayed for acetylcholine esterase activity and used for further analysis.\n\nAcetylcholine esterase assay\nA 30 μl sample of red blood cell vesicles was mixed with an equal volume of buffer (20 mM Hepes, pH 7.6, 150 mM NaCl, 0.5 % Triton X-100), vortexed and incubated for 5 minutes at 37°C. Subsequently 640 μl sodium phosphate buffer (100 mM Na2PO4, pH 7.6), 50 μl DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), 10 mM in sodium phosphate buffer), and 50 μl acetylthiocholine chloride (12,5 mM in H2O) were added. The reaction took place at room temperature and was measured photometrically at a wavelength of 405 nm according to the Ellmann' method [25].\n\nDetermination of the osmotic resistance of red blood cells\nA Na2HPO4/NaH2PO4 buffered NaCl gradient (pH 7.4) comprised of the following NaCl steps was used for this assay: 0.90%, 0.65%, 0.60%, 0.55%, 0.50%, 0.45%, 0.40%, 0.35%, 0.30%, 0.10%. For each step 5 ml of the gradient step solution and 50 μl blood sample were carefully inverted four times and incubated for 30 minutes in the dark. Samples were carefully inverted again and centrifuged at 1,500 × g for 10 minutes. From 1 ml supernatant of each step the extinction at 546 nm wavelength was measured. The experiment was performed at room temperature.\n\nPlatelet aggregation assay\nApproximately 500 μl of murine blood were centrifuged at 200 × g for 15 minutes. The resulting supernatants were transferred to new tubes and used as platelet rich plasma (PRP). The platelet aggregation measurements were done according to the method developed by Born and Cross [26] using an APACT photometer (Labor, Ahrendsburg, FRG) and the APACT software (APACT 1.4). Subsequently samples of the PRP were adjusted with buffer (5 mM Hepes, 150 mM NaCl, pH 7.3) to 250,000 platelets/μl. A constant platelet count was used throughout allowing comparisons between wild type and knock out murine and human samples. 250 μl adjusted platelets were prewarmed to 37°C and used in the assay, and the aggregation process was started by adding 25 μl of a prewarmed 16.5 mg/ml ristocetin solution (DiaMed Diagnostica, Bensheim, FRG; concentration in the assay: 1.5 mg/ml), a reagent which worked reliably in our hands with mouse and human platelets. Each murine assay represents a different animal. Analyses of human blood samples were performed as described in the manufacturer's protocol. Several repeated measurements were done per human individual to control the reproducibility. The acquired aggregation curve data, measured as ascending degree of transmission, were analysed for maximal slope values, while the maximal aggregation amplitude of every single curve was set to 100 %. In general, all platelet procedures were done at 22°C within four hours after collecting the blood sample.\n\nMiscellaneous methods\nFor standard haematopoetic measurements and cell counts on murine and human blood samples about 300 μl of blood were analysed with an ADVIA 120 electronic cell counter (Bayer). With this equipment red blood cells and platelets are \"transformed\" into spherical bodies without changing their volume. Therefore number and volume of red blood cells and platelets are not calculated values, but directly measured values. The following data were statistically analysed: leucocytes, red blood cells, haemoglobin, haematocrit, mean cellular volume, mean cellular haemoglobin, mean cellular haemoglobin concentration, platelet number, neutrophiles, lymphocytes, monocytes, eosinophiles, basophiles.\nPeptide mass fingerprinting was performed on a Bruker Reflex IV MALDI-TOF mass spectrometer. Recombinant annexin A7 was purified according to [27], F-actin-binding of recombinant annexin A7 was done as described [28]. SDS-PAGE and western blotting were done as described [29,30]. Detection in immunoblots was with enhanced chemiluminescence [17]. Antibodies employed were mab 203–217 directed against mouse annexin A7 core domain [5] and a polyclonal rabbit anti-sorcin antibody [31].\nFor statistical analyses the following tests were performed: David et al. range test to ensure samples are from a normal distribution, F-test looking for a samples' homoscedascity, Student's T-test to judge if samples of different groups have a different mean value and are parts from different normal distributions. The exact probability values and the significance of an analysis are indicated when the experiments are described."}

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craft-sa-dev

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craft-ca-core-ex-dev

PMC:194730 / 27099-35577 JSON TXT