Sample Collection and Storage
Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 15 minutes at 1000×g at 2 - 8℃. Collect the supernatant to carry out the assay. Blood collection tubes should be disposable, non-endotoxin.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8℃ within 30 minutes of collection. Collect the supernatant to carry out the assay. Hemolysis samples are not suitable for ELISA or CLIA assay.
Cell lysates: For adherent cells, gently wash the cells with moderate amount of pre-cooled PBS and dissociate the cells by trypsin. Collect the cell suspension into the centrifugal tube and centrifuge for 5 minutes at 1000×g. Discard the medium and wash the cells for 3 times with pre-cooled PBS. For each 1x106 cells, add 150-250uL of pre-cooled PBS (0.01M, pH=7.4) to keep the cells resuspended. Repeat the freeze-thaw process for several times until the cells are lysed fully. Centrifuge for 10minutes at 1500×g at 2 - 8℃. Remove the cell fragments, collect the supernatant to carry out the assay. Avoid repeated freeze-thaw cycle.
Tissue homogenates: You’d better get detailed references from other literatures before assay aiming at different tissue types. For general information, hemolysis blood may affect the result, so you should mince the tissues to small pieces and rinse them in ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (tissue weight (g): PBS volume (mL) =1:9) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5 minutes at 5000×g at 2 - 8℃, collect the supernatant to carry out the assay.
Cell culture supernatant: Collect samples and centrifuge for 20 minutes at 1000×g at 2 - 8℃. Collect the supernatant to carry out the assay.
Saliva: Remove particulates by centrifugation for 10 minutes at 4000×g at 2-8°C. Collect the supernatant to carry out the assay. Recommend to use fresh saliva samples.
Urine: Use a sterile container to collect urine samples. Remove particulates by centrifugation for 15 minutes at 1000×g at 2-8°C. Collect the supernatant to carry out the assay.
Stool: Collect feces, suspend the stool with PBS (0.01M, pH=7.4), oscillate on the ice for 15 minutes and then centrifuge for 5 minutes at 5000×g at 2 - 8℃. Collect the supernatant to carry out the assay.
Other biological fluids: Please contact tech-support for details.
Note for sample:
- Samples should be assayed within 7 days when stored at 4℃, otherwise samples must be divided up and stored at -20℃ (≤1 month) or -80℃ (≤3 months). Avoid repeated freeze-thaw cycles. Centrifuge again before assaying to remove any additional precipitates that may appear after storage.
- Please predict the concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
- If the sample type is not included in the manual, a preliminary experiment is suggested to verify the validity.
- If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance.