Use the data to solve the multistep question. Please provide clear answers to the questions.

Optical Density Readings (540nm) replicate 1 replicate 2 replicate 1 replicate 2 replicate 3 replicate 1 replicate 2 replicate 3 STANDARD stimulation conditon A stimulation conditon B 0.046 0.032 0.030 0.035 0.033 0.031 0.032 known concentrations of TNFa (pg/mL) for standard curve 700 350 175 87.5 43.71 21.8 10.9 0 0.047 0.035 0.034 0.038 0.043 0.031 0.027 0.038 0.052 0.053 0.039 0.042 0.036 0.032 0.029 stimulation conditon A (10x dilution) 0.048 0.034 0.045 stimulation conditon B (10x dilution) 0.066 0.042 0.055 BLANK 0.029 0.051 0.029 0.029 0.029 + control 0.029 - control Optical Density Readings (450nm) replicate 1 replicate 2 replicate 1 replicate 2 replicate 3 replicate 1 replicate 2 replicate 3 STANDARD stimulation conditon A stimulation conditon B 1.0190 1.1120 2.310 1.790 2.430 1.879 1.341 1.720 0.5470 0.5300 0.2700 0.2680 0.1370 0.1490 stimulation conditon A (10x dilution) stimulation conditon B (10x dilution) 0.1050 0.0960 0.220 0.199 0.226 0.137 0.152 0.146 0.0670 0.0700 0.0510 0.0510 0.0350 0.0370 0.379 0.376 0.356 0.047 0.068 0.055 + control - control BLANK Subtract the background readings taken at 540nm from the experimental readings taken at 450nm for each well. This value is your correction for optical imperfections in the plate. Average the above calculated, optically corrected replicate readings for wells containing your 0 pg/mL (BLANK) standard. Subtract the average value of the blank calculated in step #2 from all of your corrected standard and experimental samples. This is your background subtraction. Average replicate readings for all groups. Create a standard curve by plotting the known TNFa concentration of each on the x-axis against the corrected, averaged measured absorbance (optical density) for each standard on the y-axis. Draw a best fit line through the points on the graph of your standard curve to obtain your equation for the line of best fit (hint: y = mx +b, you can use the software to do this), and R^2 value. Paste a copy of your graph in this file, and record your: R^2 value = equation for the line of best fit, What does the R^2 value tell you about the standard curve performed in this assay? To calculate the TNFa concentration of each experimental group, plug the measured optical density value (y) into your best fit equation and solve for x (TNFa concentration). Complete the table with the final calculated concentration of TNFa for each stimulation condition (remember to account for indicated dilution factors of the sample!). pg/mL EXPERIMENTAL GROUP stimulation condition A stimulation condition B stimulation condition A (10x dilution) stimulation condition B (10x dilution) positive control negative control Can you trust that the TNFa pg/mL OD value obtained in wells corresponding to experimental conditions A and B is accurate? Why or why not? Can you trust that the TNFa pg/mL OD value obtained in wells corresponding to experimental conditions A and B (10x dilutions) is accurate? Why or why not? Optical Density Readings (540nm) replicate 1 replicate 2 replicate 1 replicate 2 replicate 3 replicate 1 replicate 2 replicate 3 STANDARD stimulation conditon A stimulation conditon B 0.046 0.032 0.030 0.035 0.033 0.031 0.032 known concentrations of TNFa (pg/mL) for standard curve 700 350 175 87.5 43.71 21.8 10.9 0 0.047 0.035 0.034 0.038 0.043 0.031 0.027 0.038 0.052 0.053 0.039 0.042 0.036 0.032 0.029 stimulation conditon A (10x dilution) 0.048 0.034 0.045 stimulation conditon B (10x dilution) 0.066 0.042 0.055 BLANK 0.029 0.051 0.029 0.029 0.029 + control 0.029 - control Optical Density Readings (450nm) replicate 1 replicate 2 replicate 1 replicate 2 replicate 3 replicate 1 replicate 2 replicate 3 STANDARD stimulation conditon A stimulation conditon B 1.0190 1.1120 2.310 1.790 2.430 1.879 1.341 1.720 0.5470 0.5300 0.2700 0.2680 0.1370 0.1490 stimulation conditon A (10x dilution) stimulation conditon B (10x dilution) 0.1050 0.0960 0.220 0.199 0.226 0.137 0.152 0.146 0.0670 0.0700 0.0510 0.0510 0.0350 0.0370 0.379 0.376 0.356 0.047 0.068 0.055 + control - control BLANK Subtract the background readings taken at 540nm from the experimental readings taken at 450nm for each well. This value is your correction for optical imperfections in the plate. Average the above calculated, optically corrected replicate readings for wells containing your 0 pg/mL (BLANK) standard. Subtract the average value of the blank calculated in step #2 from all of your corrected standard and experimental samples. This is your background subtraction. Average replicate readings for all groups. Create a standard curve by plotting the known TNFa concentration of each on the x-axis against the corrected, averaged measured absorbance (optical density) for each standard on the y-axis. Draw a best fit line through the points on the graph of your standard curve to obtain your equation for the line of best fit (hint: y = mx +b, you can use the software to do this), and R^2 value. Paste a copy of your graph in this file, and record your: R^2 value = equation for the line of best fit, What does the R^2 value tell you about the standard curve performed in this assay? To calculate the TNFa concentration of each experimental group, plug the measured optical density value (y) into your best fit equation and solve for x (TNFa concentration). Complete the table with the final calculated concentration of TNFa for each stimulation condition (remember to account for indicated dilution factors of the sample!). pg/mL EXPERIMENTAL GROUP stimulation condition A stimulation condition B stimulation condition A (10x dilution) stimulation condition B (10x dilution) positive control negative control Can you trust that the TNFa pg/mL OD value obtained in wells corresponding to experimental conditions A and B is accurate? Why or why not? Can you trust that the TNFa pg/mL OD value obtained in wells corresponding to experimental conditions A and B (10x dilutions) is accurate? Why or why not