Note: In case follow-up experiments are needed, it is strongly recommended to aliquot all samples after preparation to minimize cytokine degradation from multiple freeze-thaw cycles.
How do I prepare plasma and serum samples?
1. Collect whole blood into an EDTA, Citrate or Sodium heparin tube (e.g. BD vacutainer, Cat. No: 8001302 or 16852). 2. Centrifuge 10 minutes at 3,000 rpm. 3. Take supernatant and aliquot into small tubes and store at -80°C until use.
1. Collect whole blood into a tube without additives (e.g. BD vacutainer, Cat. No. 8002527). 2. Keep at room temperature for 20 minutes. 3. Centrifuge 10 minutes at 3,000 rpm. 4. Take supernatant and aliquot into small tubes and store at -80°C until use.
How do I prepare urine samples?
1. Collect urine without adding stabilizers. 2. Centrifuge the samples hard (eg. 10,000 x g for 1 min or 5,000 x g for 2 min). 3. Aliquot, quick freeze in dry ice, and store at -80°C until use.
How do I prepare conditioned media samples?
We recommend preparing serum-free or low-serum medium samples, as serum tends to contain cytokines which may produce significant background signals. If it is necessary to test serum containing medium, we recommend also running an uncultured media blank to assess baseline signals. This baseline can then be subtracted from the cultured media sample data.
1. On day 0, seed ~1 million cells in 100 mm tissue culture plate with complete medium.* 2. On day 3, remove medium and replace medium with 6-8 ml of serum-free or low serum containing medium (e.g. medium containing 0.2% calf serum). 3. On day 5, collect medium into 15 ml tube. Centrifuge at 2,000 rpm in centrifuge at 4ºC for 10 minutes. Save the supernatant. Transfer the supernatant into 1.5 ml Eppendorf tubes. Store supernatant at -80ºC until experiment. Most samples can be stored this way for at least a year. *The optimal number of seeded cells varies from one cell type to another and may need to be empirically determined.
How do I prepare cell or tissue lysate samples?
1. Pre-cool a refrigerated centrifuge to 4°C. Pellet the cultured cells by centrifugation for 5 minutes at 1000 x g (approximately 2000 rpm) at 4°C. Wash 3 times with ice-cold 1X PBS and then add chilled RIP buffer with protease inhibitor. In general, add 100 μl RIPA buffer for approximately every 106 cells present in the pellet (count cells before centrifugation). Reduce the volume of RIPA buffer accordingly if a higher protein concentration is required. Vortex to mix and keep on ice for 30 min, vortexing occasionally. Go to step 3, lysis and storage.
2. Dissect the tissue of interest and wash briefly with chilled 1X PBS to remove any blood if necessary, cut the tissue into smaller pieces whilst keeping it on ice. Transfer the tissue to a homogenizer and add RIPA buffer with protease inhibitor. In general, add 500 μl RIPA buffer for approximately every 10 mg of tissue. Homogenize thoroughly and keep the sample on ice for 30 min. Vortex occasionally. Go to step 3, lysis and storage.
Lysis and Storage
3. Sonicate the sample to break the cells or tissue up further and to shear DNA. Adjust sonication time to your type of sample: 1 min for cell lysates and 2-5 min for tissue lysates at a power of about 180 watts (in rounds of 10 seconds sonication/10 seconds rest for each cycle). Keep the sample on ice during the sonication. 4. Centrifuge at 10,000 x g (approximately 9700 rpm for rotors of a 9.5 cm radius) for 20 minutes at 4°C to pellet cell debris, and then transfer the supernatant to a fresh microfuge tube without disturbing the pellet. 5. Determine protein concentration of the lysate by Bradford or BCA protein assay. 6. Aliquot samples and freeze at -80°C.
RIPA buffer (1000ml)
50 mM Tris•HCl, pH 7.4 - 50ml 150 mM NaCl - 8.76g 1% Triton X-100 or NP-40 - 10ml 0.5% Sodium deoxylcholate - 5g 0.1 % SDS - 1g 1 mM EDTA (0.5 M stock) - 2ml 10 mM NaF - 0.42g
Add ddH2O to 1000 ml; Add PMSF to a final concentration of 1 mM and any other protease inhibitors immediately before use.
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