生物活性

• 描述Sulforhodamine B is a dye used for cell density determination, based on the measurement of cellular protein content.You may follow this protocol (from https://www.ncbi.nlm.nih.gov/pubmed/28573164) A. Treatment solution preparation Volumes of treatment of choice should be enough for triplicates in 96-well plates (50 μl per replicate; final volume in well 100 μl) or six replicates in 384-well plates (10 μl per replicate; final volume in well 20 μl) and also account for pipetting variation. Treatment can be prepared in aqueous solution (Opti-MEM for transfections) or solvent of choice (e.g., DMSO). B. Cell preparation Remove medium from cell monolayers and wash the cells once with sterilized PBS. Remove PBS and add 1 ml (100 mm plates) 0.25% (wt/vol) trypsin to evenly cover the cell-growth surface. Incubate at 37 °C for 5 min or until cells start to dissociate. Next inactivate trypsin with 10 volumes of culture medium containing FBS, and mix up and down to obtain a homogeneous single cell suspension. Transfer the cell suspension to a sterile Falcon tube. Determine the cell concentration by counting in a hematocytometer chamber under a microscope using a 1:1 mixture of cell suspension and 0.4% (wt/vol) trypan blue solution to determine cell viability prior cell seeding. Optional: before counting, spin down cells in order to wash trypsin and resuspend in growth medium. Adjust the cell concentration with growth medium (10% FBS) to obtain an appropriate cell seeding density per well in a volume of 50 μl (96-well format) or 10 μl (384-well format). Note: Initial cell seeding density will depend on two main factors: 1) doubling time of the cell line used and 2) the day the plate will be read. Cells are typically at 70–80% confluency (2 × 104 for 96-well format and 8 × 103 for 384-well format) by the end of the experiment (day 5). Transfer the cell suspension into a sterile reagent reservoir to make it easier to pipette with a multichannel pipette. C. Treatment exposure Mix the treatment solutions prepared in step A by pipetting. Dispense 50 μl (96-well format) or 10 μl (384-well format) of solution into each well. Mix cell suspension prepared in step B thoroughly and add 50 μl (96-well format) or 10 μl (384-well format) to each well already containing treatment solutions. Note: Ensure even cell distribution in the bottom of the plate and avoid shaking the plate to avoid 'ring effects'. The best way to achieve this is to add the cell solution directly to the bottom of the well, avoiding touching the walls. We also recommend performing a short spin of the plate (20 sec, 10 × g) before placing it in the incubator. Set aside three wells in the plate containing only Opti-MEM or solvent of choice and cell suspension for an untreated or vehicle control. Also, leave three wells in the plate containing only medium for background subtraction. Incubate the plate at 37 °C in a humidified incubator with 5% CO2 until plate is to be read. Note: The incubation time will depend on the type of compound being tested and has to be experimentally determined. For example, in our hands we observe the biggest difference in cell proliferation between negative control and a tumor suppressive miRNA 5 days following transfection. D. Cell fixation and staining Gently add 25 μl (96-well format) or 5 μl (384-well format) cold 50% (wt/vol) TCA to each well directly to medium supernatant, and incubate the plates at 4 °C for 1 h. Mixing is not required, as this could lead to some cells detaching from the bottom of the well. Wash the plates four times by submerging the plate in a tub with slow-running tap water and remove excess water by gently tapping the plate into a paper towel. After the last wash allow the plate to air-dry at room temperature. Note: Cell monolayer detachment can occur if water is forced into the wells. We recommend letting the plate dry completely before continuing to next step. If necessary, dried plates can be stored at room temperature indefinitely. Add 50 μl (96-well format) or 20 μl (384-well format) of 0.04% (wt/vol) SRB solution to each well. Note: Ensure that the solution is in direct contact with the bottom of the well and that there are no bubbles in between. SRB solution is not light sensitive thus plate does not need to be covered during incubation. Leave at room temperature for 1 h and then quickly rinse the plates four times with 1% (vol/vol) acetic acid (200 μl for 96-well format or 30 μl for 384-well format) to remove unbound dye. Allow the plate to air-dry at room temperature. Note: Non-homogeneous washes are a major source of error in this assay. Washes must be done quickly and homogeneously across the entire plate. This is particularly important for 384-well format. The small area of the wells leads to high superficial tension sometimes making it difficult to wash the plates uniformly. Visually check that all the wells in the plate have been injected with 1% (vol/vol) acetic acid and that there are no bubbles preventing the washes. We recommend washing with multi-channel pipet and injecting the solution indirectly into the wells using the walls of the wells. E. Absorbance measurement Add 50 μl to 100 μl of 10 mM Tris base solution (pH 10.5) to each well and shake the plate on an orbital shaker for 10 min to solubilize the protein-bound dye (approximately 10 min). Measure the absorbance at 510 nm in a microplate reader^[2].