Hello Erjola Rapushi

You could use the following protocol.

1. Seed cells in 96-well plate at an appropriate cell density.

2. Treat cells with the drug candidate at the desired drug concentrations once the cells have attached to the substratum.

3. After the treatment period, withdraw culture medium from each well.

4. Rinse the cells by carefully adding 200μL PBS per well and aspirate to waste. Add 100μL cell lysis buffer to each well and place the plate on a shaking platform (at low speed for 10-20 minutes). Please note: The lysis buffer should be supplemented with protease inhibitors at the time of lysis to minimize protein degradation upon release of cellular proteases.

6. Agitate the liquid in each well by pipetting up and down and transfer the cell lysates to 0.5 mL microcentrifuge tubes.

7. Centrifuge the samples at 14,000 × g for 5 minutes to remove cell debris.

8. Transfer supernatants to clean tubes. You could either proceed to the next step or store the cell lysates at –20 °C (for 1 month) to perform the assay later.

9. The protein concentration of your lysates may be determined by a total protein assay such as the Bicinchoninic acid assay. The volume of each sample can also be normalized to deliver the same amount of total protein for each assay.

10. You could perform the micro-BCA assay as follows.

You may add 5μL of sample to a 96-well microplate followed by 100μL of BCA reagent. Mix plate thoroughly on a plate shaker for 30 seconds. The plate may be incubated in the dark for 30min at 37 °C. The absorbance may be measured at 560nm, and protein concentration may be determined from a BSA standard curve.

11. Then perform ELISA for cytokines.

Best.

Hello Eroja, We use a bead system eg CBA to detect cytokines by flow cytometry but essentially the same process. Cells are separated from diluent by centrifugation in a plate spinning centrifuge @ 300g for 10 minutes, removing the diluent for subsequent labelling. Resuspend the pellets on a plate shaker and wash with 2-3 cycles of 200µl fresh media/spinning. Then pellets resuspended phenotyped and/or lysed eg freezing and supernatant evaluated. Alternatively, there are plate based system eg LEGEND MAX for many cytokines.

Best wishes