Serum tube additives

Serum tubes (with additives) Thrombin (dry additive) Gray/yellow Orange... 

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

X 10 2 M Tris HC1 buffer, pH 7.5, 2.4 X 10 3 M glucose 6-phosphate (G-6-P), 1.6 units glucose 6-phosphate dehydrogenase (G-6-P dH), 5.1 X 10"5 M NADP, and 2.7 X 10 3 M KC1. In addition, the following chemicals were included in the final concentration indicated 5.1 X 10-3 M NADH, 1% (W/V) bovine serum albumin (BSA), and 1.0 mg aldrin in 0.1 ml ethanol. Whole body homogenate experiments included all of the above chemicals unless otherwise noted. Reaction mixtures were incubated with swirling in test tubes at 30 - 1°C. Reactions in Steps 1-4 of the experimental sequence were stopped after 1 hr and Steps 6-8 after 15 min, by the addition of 2 ml 5% TCA. 

Because wortmannin is hydrophobic (on the day of the experiment we dissolve an aliquot in DMSO and never keep it for more than a day or two, away from light) and used at very low concentrations, it should not be mixed with serum or other protein-rich media too long before the experiment. The DMSO solution should be added to the medium immediately before addition to the cells. If left in aqueous medium too long before addition, it will bind to the walls of the tube. 

The dry matrix contains additives that need to be removed by these washing steps. Avoid mechanical stirring of the gel, since this cannot only damage the gel matrix, but can also lead to denaturation of the immunoglobulins. A convenient way to keep the matrix gently suspended is by placing the tube on a nutator or serum incubator. 

The procedure will be described for the analysis of two different serum samples. Turn on the spectrometer and allow warm-up for 15 to 20 minutes. Set the wavelength to 510 nm. Obtain two 3-mL cuvettes or colorimeter tubes for the blank and standard. In addition, one cuvette will be needed for each serum sample to be tested. Label the cuvettes Blank, Standard, Serump and Serum2. Make the following additions ... 

Method A. The rationale of method A is that HDL and LDL are separated by selective precipitation of LDL by dextran sulfate and Mg2+ after the reaction between LDL and reconstituted HDL containing radiolabeled CE by CETP. The method was originally described by Kato et al. [77], The assay mixtures consist of reconstituted [14C]CE-HDL as the donor for CE, LDL as the acceptor, 5,5 -dithiobis-2-nitrobenzoic acid, bovine serum albumin (BSA), partially purified CETP, and a test sample in Eppendorf tubes (1.5 ml). After a 30-min incubation at 37°C, the reaction is terminated by the addition of an LDL-precipitation solution. After standing for 20 min in an ice bath, the assay mixtures are centrifuged, and the supernatant solution containing [14C]CE-HDL is analyzed for radioactivity. Furthermore, the [14C]CE-LDL precipitate is also analyzed for radioactivity if necessary. Usually the blank and control transfer values are about 6% and 34% of initial [14C]CE-HDL added under the assay conditions, respectively. 

Methyl derivatives were used by Martin and Driscoll [509] they were prepared by treatment with dimethyl sulphate and applied to the analysis of barbiturates in serum, as follows. A 2-ml volume of a saturated solution of sodium dihydrophosphate and 10 ml of diethyl ether were introduced into a test-tube containing 2 ml of serum. The test-tube was closed and shaken vigorously for 30 sec, centrifuged for 1 min, then 7 ml of the extract were transferred into another test-tube and the ether was removed in a stream of air in a water-bath. The residue was dissolved in 2 ml of methanol with 10% of water (v/v), saturated with potassium carbonate, and 0.1 ml of dimethyl sulphate were added and the mixture was heated in a water-bath at 60°C for 4 min. At this temperature methanol was removed in a stream of air (3 min) and the residue was extracted into 1.5 ml of -heptane with the addition of 1 ml of 1 M acetate buffer (pH 6). A 1 -ml volume of the n-heptane extract was transferred into a small test-tube, evaporated carefully in order that losses of the derivatives might be avoided, and dissolved in 100 (A of acetone (containing an internal standard if necessary) 5 jul were analysed on a column packed with 5% of SE-30 at 175°C. 

The reaction mixture contained 100 /xL of serum sample 400 /xL of 0.8 mM cytidine in 50 mM potassium phosphate buffer (pH 7.0). The tubes were capped and incubated for 20 minutes at 56°C. Aliquots of 400 /u.L were then removed and added to 100 /xL of cold 6% (v/v) perchloric arid containing 0.75 mM allopurinol. Mixing was followed by addition of 500 /xL of 100 mM ammonium acetate (pH 7.0). The supemate obtained by centrifugation was then analyzed by HPLC. 

Procedure. Acetate buffer (450 p. ), DNA (100 /u.1), KI (100 /xl), and HjO (150 /Lil), are added to a 12 x 125 mm Pyrex glass test tube, which is then sealed with a 13 x 20 mm serum bottle stopper. All further additions and withdrawals are made through this stopper by means of hypodermic syringes equipped with a 25-gauge needle 5 ml of air are withdrawn, and... 

Insulin. Twelve glass test tubes are labeled, and 0.2 ml of phosphate buffer 1 is added to each tube. The two blank tubes receive 0.1 ml of phosphate buffer 2, and the remaining 10 tubes receive in duplicate 0.1 ml of five increasing dilutions of anti-insulin serum in phosphate buffer 2. A constant amount (0.1 ml) of [ I]insulin in phosphate buffer 1 is added to each tube, and the tubes are incubated at 37° for 45 min. The reaction is stopped by the addition to each tube of 5 ml of the Z-gel slurry followed by 10 ml of 0.1 Af NH Ac (pH 6.25). The contents of the tubes are mixed by inversion, and the Z-gel is collected by centrifugation (1000 g for 5 min) at room temperature. The supernatant is decanted, and the pellet is washed by resuspension in 10 ml of 0.1 M NH4AC followed by recentrifugation. The supernatant is decanted, and the amount of associated with the pellet of Z-gel is measured. Approximately 80% of the I in the preparation of [ I]insulin binds to Z-gel in the presence of excess antibody, but only 10% binds in the absence of antibody. A dilution of the... 

Carcinoembryonic Antigen Fig. lA). Five mililiters of EDTA buffer is added to each of 10 glass test tubes and then 10, 25, 50, and 100 pi (1.25, 3.125, 6.25, and 12.5 ng) of the CEA standard are added in duplicates to 8 of the tubes. Two tubes receive no nonradioactive CEA. Twenty-five microliters of the appropriate dilution (see titration curve) of anti-CEA serum are added to each tube the tube contents are mixed and then incubated in a H2O bath for 30 min at 45°. After this initial incubation period, the tubes are removed from the bath, and 25 p of I-labeled CEA are added to each tube. The tubes are returned to the 45° bath and are incubated for an additional 30 min. The tubes are removed from the bath, Z-gel is added to stop the reaction, and the tubes are processed as described for the titration curve. 

Insulin Fig. 1C). Standard inhibition curves are prepared using 10 tubes, each of which contains 0.1 ml of buffer 2 and 0.1 ml of the appropriate dilution of anti-insulin serum. Four pairs of tubes receive 0.1 ml of the insulin standard diluted so as to contain 1.0, 2.5, 5.0, or 10.0 /alU of insulin. The tubes are incubated at 37° for 2 hr, and then 0.1 ml of P I]in-sulin is added to each. After an additional incubation period of 45 min at 37°, 5 ml of Z-gel are added and the tubes are processed as described (see titration curve). 

The antibody-coated red cells are prepared as previously described. It is particularly important for this procedure that the indicator red cells are absolutely free of clumps. The sensitivity of coated cells can be assayed by reverse passive hemagglutination if, as in the model under consideration, the antigen is available in soluble form. The cells under study are washed in suitable tissue culture medium or other buffered solution and suspended at a concentration of 10 per milliliter in the same diluent to which serum has been added (usually 1% fetal calf serum). A small volume (50-100 /U.1) of the cell suspension is placed in a 10 x 75 mm disposable tube. The addition of an equal volume of 1% coated red cells results in a mixture that contains about 25 red cells per lymphocyte. Linkage of antibody on the red cells to the corresponding antigen determinant on the surface of the lymphocyte results in the formation of a rosette or lymphocyte surrounded by red cells. The mixing of cells and incubation for at least 1 hr are done in an ice bath. The tubes are then centrifuged very briefly (1 min at 1000 g), and a drop of dye is added to tint the lymphocytes (e.g., crystal violet or brilliant cresyl blue). The mixture is then aspirated four or five times with a Pasteur pipette and examined in a hemacytometer chamber at about 400 x. A cell is scored as a rosette if it is surrounded by three or more adherent erythrocytes, and usually 300 cells are counted. 

Willis (Wll), using a potassium hollow cathode tube instead of the commonly employed discharge lamp, determined potassium in blood serum. At the 1 50 dilution no interference was encountered from calcium, magnesium, and phosphate at serum levels, but sodium gave a small enhancement. The sodium interference was controlled by the addition in excess of sodium chloride or of the disodium salt of EDTA to samples and standards alike. 

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