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- EXPERIMENT: CRYOPRESERVATION OF CULTURED CELL LINES Procedure for Cryopreserving MCF-7 and THP-1 Cells• Ensure that the culture is up to the late log phase (assumption is made based on the cell concentration). Prepare the cell suspension and determine the cell concentration.• Dilute the cell suspension in 1:1 ratio with freezing medium (final concentration approximately 1 X 106 – 1 X 107cells/ml is desired). • Dispense the cell suspension into prelabeled cryovial and place the vial in -80°C freezer overnight (temporary storage).• Transfer the cryovial into liquid nitrogen cryotank for long term storage the following day.• After day 3, determine the cell concentration in the freezing medium andcompare with that of the stock concentration. Question: Please briefly explain what is cryopreservation(1), how procedure above are useful to make this experiment successful(2)Using two centrifuges (A and B), with maximumspeeds of 1500 rpm (r = 8 cm) and 3000 rpm(r = 8 cm), respectively, construct a separationscheme for separating three subcellular structureswith the following xg requirements:• PAT – 150 xg• SAT – 120,000 xg• HAT – 650 xg24. Below, see the results of Subject A's DXA scan. In the third column, the enhanced analysis shows percent fat (% Fat) in various regions of the body. In the row for Total, the highlighted number represents the estimate of Subject A's percent body fat. According to estimates of % body fat derived from DXA, does Subject A have excess body fat?
- mixed micron cuboidal shaped cells within the stronger paste (F127) and the physical/biochemical process that occurs during the gelationHoechst 33342 is a membrane-permeant dye thatfluoresces when it binds to DNA. When a population ofcells is incubated briefly with Hoechst dye and then sortedin a flow cytometer, which measures the fluorescence ofeach cell, the cells display various levels of fluorescence asshown in Figure Q17–1.In an experiment to investigate membrane integrity, beet root sections were subjected to different chemicals. Membrane damage was estimated by the color intensity of leaked pigment. Five replicates of each treatment were prepared. Complete the following table with the absorbance values. Calculate the mean (average) and the standard Table 1: Absorbance readings of pigment leaked from damaged cells treated with two different chemicals. Replicate # Absorbance at 460 nm Chemical 2 Chemical 6 1 0.357 0.392 2 0.576 0.395 3 0.334 0.385 4 0.197 0.399 5 0.334 0.403 Average SD Calculate the Coefficient of Variance (see the appendix) for the readings of each chemical: Evaluate the precision of each and state which one is more precise:
- Please answer fast What are some similarities and differences in Bradford assay and Nanodrop (to obtain direct absorbance measurements). What are the benefits and setbacks of them. How is 260/280 absorbance ratio significant?Time point (min) Absorbance of culture at 660nm Approximate cell concentration Approximate # cells in 1mL extract 0 0.298 1.49 x 108 cells/mL 1.49 x 108 cells 10 0.316 1.58 x 108 cells/mL 1.58 x 108 cells 20 0.374 1.87 x 108 cells/mL 1.87 x 108 cells 30 0.429 2.145 x 108 cells/mL 2.145 x 108 cells 40 0.512 2.56 x 108 cells/mL 2.56 x 108 cells 50 0.544 2.72 x 108 cells/mL 2.72 x 108 cells 60 0.607 3.035 x 108 cells/mL 3.035 x 108 cells a. Using these data, prepare a growth curve of this strain ofEscherichia coli (E. coli).b. Estimate the doubling time for this strain of E. Coli. Clearly showhow you estimated this value from the empirical data presented.For the serial dilution, your stock solution must have a concentration of 3.5 mg/mL. How much diluent must be added to the 5.3 mg/mL red cell to prepare the stock solution? Show pertinent solution/s. What are the initial concentrations used for tubes 3, 5, and 6? Show pertinent solutions. What are the dilutions of the last positive tube and first negative tube respectively? Show computation.
- If the volume of a Staphylococcus aureus cell is estimated at 0.5 μm3, how many cells could be accommodated, in principle, in 5 mL of saturated culture? (1 mL = 1 cm3). Show your calculations.The culture you are working has a doubling time of 2 hours and a cell density of 3 x 106 cells per mL. For your experiment, you first dilute the culture 100 fold. Assuming that there is no lab phase and that the cells remain in exponential growth the entire time, what is the cell density (cells/mL) after 10 hours?EXPERIMENT: CRYOPRESERVATION OF CULTURED CELL LINES Based on the experiment, please answer the following questions. 1. Why cryoprotectant was used to freeze the cells?2. Besides DMSO, suggest 3 other chemicals that potentially be used as cryoprotectant.3. Describe the mechanism of DMSO in shielding the cells from extreme temperature.4. In your opinion, why do we need to freeze the cells in high concentration? Justify your answer.