Sample preparation refers to a series of processing steps that are performed on a sample before an experiment or analysis is performed. These steps are designed to prepare the sample to ensure that it is suitable for the requirements of subsequent experiments or analyses. The specific steps for sample preparation can vary depending on the type of experiment and the type of sample.
There are many types of samples used in multiplex cytokine assays, including serum, plasma, cell culture supernatant, urine, and lysates. Proper sample pretreatment is the first step in ensuring the accuracy of multiplex assays. Here, Xiaobei will introduce several different common sample pre-transportation methods:
Sample type
Plasma:Blood was collected with an anticoagulant-containing blood collection tube (EDTA anticoagulant tube is recommended), the supernatant was taken after two centrifugations, and the -80 was cryopreserved after aliquoting. Avoid repeated freeze-thaw cycles and transport with dry ice.
Serum:Blood was collected with a blood collection tube without anticoagulant (which can promote coagulation or empty tube), 4 and let stand for 2 hours (to avoid shock and prevent hemolysis), and the supernatant was taken after two centrifugations, and the supernatant was frozen after aliquot-80. Avoid repeated freeze-thaw cycles and transport with dry ice.
Tissue lysate:Add an appropriate amount of lysate to the tissue (protease inhibitors and phosphatase inhibitors need to be added to the lysate, 20mg of tissue corresponds to 200-300ul lysate), treat the tissue homogenate with a tissue homogenizer, place it in the refrigerator (4, 30min), take the supernatant after two centrifugation, and measure the total protein concentration of BCA in the tissue lysate (the recommended concentration is not less than 1mg ml). After aliquoting-80 cryopreservation and shipping with dry ice.
Cell lysate:After collecting cell samples, wash with PBS, add an appropriate amount of lysate (protease inhibitors and phosphatase inhibitors need to be added to the lysate, 107 cells correspond to 200UL lysate), place in the refrigerator (4, 30min), take the supernatant after two centrifugations, measure the total protein concentration of BCA (the recommended concentration is not less than 1mg ml), freeze after aliquot-80, and transport it with dry ice.
Cell culture supernatant: After two centrifugations, the supernatant was taken, aliquoted, -80 cryosteved, and transported with dry ice.
Note: Due to the high conservation of TGF- between species and the high amount of TGF- in animal serum, serum-free medium should be used when testing cell culture supernatant samples. If animal serum has already been added to the medium, a control for baseline can be added to obtain an accurate value of TGF- in the sample.
Body fluids (alveolar lavage fluid, urine, synovial fluid, cerebrospinal fluid, sputum, saliva, etc.)Nasal lavageetc.).: After two centrifugations, the supernatant was taken, aliquoted, -80 cryosteved, and transported with dry ice.
Other samples (nasal secretions, tears, uterine fluids, ** secretions, etc.).After collecting the sample, put the cotton swab into a small test tube with "bottom removed", put it into a large test tube, immediately rinse the cotton swab with the same amount of normal saline or sample diluent (such as 100ul), take the supernatant after centrifugation (the final sample amount shall not be less than 60ul), freeze it after aliquoting -80 ul, and transport it with dry ice.
Applicable Platforms
Liquid phase microarray (Luminex).
Luminex technology is based on fluorescent-encoded microspheres as the core, integrating the principle of flow cytometry, laser analysis, high-speed digital signal processing and other technologies, multi-index parallel analysis, and can accurately and quantitatively detect up to 2-100 different biomolecules at the same time in one tube; It has the characteristics of high throughput, high sensitivity, and parallel detection; It can be used for immunoassay, nucleic acid research, enzymatic analysis, receptor, ligand recognition analysis and other research in many aspects and fields.
Electrochemiluminescence (MSD).
Electrochemiluminescence is a specific chemiluminescence reaction initiated by electrochemistry on the electrode surface, which is a perfect combination of electrochemical and chemiluminescence processes. MSD electrochemiluminescence detection uses a sulfo-tag marker that is energized on the electrode surface of the Multi-Array and Multi-SPORT microplates and then electrochemically excites the sulfo-tag marker to emit intense light.
Flow cytometry liquid phase (CBA).
The Cytometric Bead Array (CBA) is a multiplex protein quantification method based on a flow cytometry system, which can detect multiple indicators in a single sample at the same time. Flow cytometry CBA technology can detect cytokines, antibodies, antigens and other indicators of rare samples and small samples. Flow cytometry has become an indispensable technical means for basic scientific research and clinical testing due to its fast detection speed, flexible detection methods, multi-index, multi-parameter and high sensitivity.