Prelab Questions: 1) What is the purpose of using trypsin?

What can happen if the cells are left in trypsin for too long? 2) How does paraformaldehyde work, and what precautions should one take when working with it. 3) Calculate the calcium (mg/dl) in a sample with absorbance 700nm, line given by y=.0547x+.01 and dilution of 50% 4) What other methods can you think of for determining the extent of differentiation of cells? 5) What is the purpose of the standard in the calcium assay?

2.1. Splitting the cells using trypsin: Materials: 1. 10% FBS growth media 2. 0.25% Trypsin 3. Phosphate buffer solution (PBS) Protocol: 1. Warm up trypsin aliquot, PBS, and growth medium. Be careful not to leave trypsin in the warm water bath for too long (it self-digests). 2. Aspirate medium and wash the culture with PBS (washing with PBS allows you to remove serum from culture conditions the serum proteins inhibit trypsin). 3. Add 1.5mL of trypsin solution into the culture and place in 37C incubator for 5 minutes. 4. Image the cells after 5 minutes to determine their detachment from the surface. Note: Gentle tapping of the sides of the dish will aid in the cells detachment if theyre not freefloating after 5 minutes. 5. Add 3 ml of growth (or expansion) medium (the serum in expansion medium inhibits the function of trypsin) 6. Transfer them into a 15 mL tube. 7. Balance centrifuge and spin cell solution for 5 minutes at 1000 rpm to obtain a cell pellet. 8. Re-suspend the pellet in 2 mL medium 9. Count the cells using the Scepter (see 2.2) 10. Note down the cell numbers 11. Plate cells on 12-well plates (see 2.3) 2.2 Cell counting with Scepter: Protocol: 1. In a microfuge tube, dilute a small volume of the cell suspension (the one originally obtained from your instructor) 1:10 with PBS in a microfuge tube. 2. Turn on the Scepter by pressing the control button on the back of the instrument and wait for on-screen instructions to appear. 3. When prompted, attach a sensor to the end of the Scepter unit with the electrode

sensing panel facing toward the front of the instrument. 4. Depress the plunger, submerge the sensor in the cell suspension, and then slowly withdraw the cell suspension into the sensor. 5. Once counting is complete, you can click the control button and scroll to manually adjust the upper and lower gates. After gating, you will see recalculated data displaying a new concentration, average cell size and volume. Gating allows you to look only at the population of cells you are interested in. 6. Calculate the total number of live cells in 0.5 ml of the undiluted cell suspension: Total Live Cells=Live Cell Density X Volume of Cell Suspension 7. Sketch the size distribution of the cells. 2.3. Differentiation (osteogenic and myogenic) of cells: Materials: 1. 2 12-well plates 2. Osteogenic or myogenic medium Protocol: 1. Plate the cells at a density of 10,000 cells/cm2 onto a 12-well plate. Use 2 plates for each experiment, as we will be analyzing differentiation as a function time. You will be using 1 plate for next week and the other for the following week so that you can assess differentiation at two time points. 2. One each of the 12-well plates, assign 6 wells for specific differentiation media and 6 wells for growth media. 3. Add 1.5 mL of osteogenic medium into each of 6 wells 4. Culture the other 6 in growth (or expansion) medium. These wells will be used as a control for the differentiation experiments. 5. Take pictures of the cells (time, T = 0) 6. During every lab session, change medium and image the cells to obtain cell morphology (shape, size) data at various points for further analysis. Preparing cells for calcium quantification assay and staining at 1 week of differentiation Materials: 1. Paraformaldehyde (PFA) 2. 0.25% Trypsin 3. Phosphate buffer solution (PBS) 4. Cold 100mM Tris, pH 7.5 Protocol: 1. Warm up the differentiation and cell culture growth media. Remove one of your 12-well differentiation plates and change medium (making sure not to mix up differentiation and growth media wells!!). 2. Take the other of your 12-well differentiation plates out of the incubator to carry out analyses.

3. Plate for analyses: Use half of the differentiation (3 wells) and half of control (3 wells) for staining and the other half for calcium quantification. Wells for staining 1. Wells for staining: Remove medium and add 1mL of the 4% paraformaldehyde (PFA) solution (provided) into each well and let sit room temp for 15 minutes. BE CAREFUL NOT TO BREATHE OR COME INTO CONTACT WITH PFA, IT IS VERY CAUSTIC! 2. Gently remove the PFA and dispose as hazardous waste. 3. Add PBS into the wells 4. Wells for staining: Stain the wells using the protocol provided below. 5. Image each well using the inverted microscope. It is important to image each well, acquiring multiple images/well. Wells for calcium quantification: 1. Warm up trypsin aliquot and PBS. Be careful not to leave trypsin in the warm water bath for too long (it self-digests). 2. Aspirate medium and wash the culture with PBS (washing with PBS allows you to remove serum from culture conditions the serum proteins inhibit trypsin). 3. Add 500uL of trypsin solution into each of the calcium quantification wells and place in 37C incubator for 5 minutes. 4. Add 1 ml of PBS to each well (PBS inhibits the function of trypsin) 5. Transfer them into a 2 mL microcentrifuge tube. 6. Balance centrifuge and spin cell solution for 5 minutes at 1000 rpm to obtain a cell pellet. 7. Aspirate the supernatant. DO NOT ASPIRATE PELLET 8. Homogenize the cell pellet with 1mL of cold 100mM Tris, vortex to lyse cells 9. Centrifuge cells at 10,000 rpm for 15 minutes at 4C 10. Collect supernatant in a 1.5 mL microcentrifuge tube and store on ice Calcium Quantification Assay Materials (From Assay kit): 1. Calcium Assay Buffer (10X) 2. Calcium Standard 3. Calcium Detector R1 4. Calcium Detector R2 Protocol: Reagent Preparation 1. Add 5mL of concentrated Assay BUffer to 45 mL of HPLC grade water, will be used for diluting samples and calcium standard 2. Prepare the the calcium standard in six 200uL PCR tubes following the table below:

Standard Number 1 2 3 4 5 6

Calcium Standard (uL) Stock concentration (20 mg/dl) 0 10 25 50 75 100

Assay Buffer (uL)

Final Calcium Concentratio n (mg/dl) 0 2 5 10 15 20

100 90 75 50 25 0

3. Prepare the Working Detector Reagent by combining equal parts of Calcium Detectors R1 and R2. 5mL of each reagent will be sufficient for one plate. As 100 uL is needed per well (96 wells x 100uL = 9.6mL). Once mixed the working detector reagent is stable for 60 minutes. Performing the Assay Plate Layout: 1 A B C D E F G H S1 #1 #5 #9 #13 #17 #21 #25 2 S1 #1 #5 #9 #13 #17 #21 #25 3 S2 #1 #5 #9 #13 #17 #21 #25 4 S2 #2 #6 #10 #14 #18 #22 #26 5 S3 #2 #6 #10 #14 #18 #22 #26 6 S3 #2 #6 #10 #14 #18 #22 #26 7 S4 #3 #7 #11 #15 #19 #23 #27 8 S4 #3 #7 #11 #15 #19 #23 #27 9 S5 #3 #7 #11 #15 #19 #23 #27 10 S5 #4 #8 #12 #16 #20 #24 #28 11 S6 #4 #8 #12 #16 #20 #24 #28 12 S6 #4 #8 #12 #16 #20 #24 #28

S1-6 = Calcium Standard Wells #1-28 = Sample Wells (up to 28 samples in triplicate can be performed per plate) 1. Standard Wells: add 10uL of the calcium standard per well in the corresponding well as shown in the plate layout 2. Samples Wells: add 10 uL of sample to three wells per sample

3. Add 100uL of the working detector reagent to each well with a sample in it 4. Gently sake the plate for about 20-30 seconds 5. Incubate at room temperature for five minutes 6. Read the absorbance at 570-590nm. The resulting color is stable for at least 30 minutes Calculations: 1. Calculate the average absorbance of each standard 2. Subtract the average absorbance from Standard S1 from itself and all other values on the plate. This makes the Standard S1 samples the reference (blank/zero) and each sample will have corrected absorbance value. 3. Plot the corrected average absorbances for the standard as a function of concentration in Excel for a standard curve. 4. Calculate the calcium concentration of the samples by using the formula: Calcium (mg/dl) = Corrected Sample Absorbance - (y-intercept) Slope x Sample Dilution

Post Lab: 1) Compare your calcium calculations to the metrics obtained through imageJ. Do the two agree?

The objective of our test is to make sure our design of a new alternative module for the BENG 162 lab class is simple to read, yet efficient with enough detail. The end goal is for all students, faculty, and staff to read our module and come up with a single interpretation of the text which will eliminate confusion and produce a quality protocol which will have little to no room for misunderstandings. The test will provide a clear representation of the quality of our module design and should hit several key aspects which are a proper grammatical layout of the text, consistent interpretation of each step, reliability of the results, and the reproducibility of the module. The treatment groups involved in our tests is generally limited to ABET/BTEC students and departmental faculty because the protocol will have specific information based on laboratory equipment, procedures, and basic biological processes so anyone who reads our protocol should already have an understanding of these concepts. Any BTEC students who have already taken this class can serve as controls because they possess information on how the labs are organized so the testing group will generally be ABET students who have never taken the lab to test if they adequately understand the protocol and how to proceed through each step. Theoretically, the sample size should be as large as possible so the results are as accurate as possible, but due to lack of time, there will likely only be time for roughly 50 tests to be administered. The testing will be done on an individual basis and done by appointments to avoid time conflicts so the ability to do massive wide screening of tests is limited and will not be a possibility unless there is more time. Besides that point, a sample size of 50 will provide adequate information on the reproducibility and interpretation aspects of our goals, but the reliability of the results will require more samples to be administered for us say that our protocol reliably achieves a certain result without a doubt. The statistical tests to be used for the quality assurance of our protocol will be a quickly devised multiple choice exam which will hit key aspects of our protocol ranging from general knowledge to more detailed aspects of our protocol. This multiple choice exam will provide insight into how easy it was to interpret our protocol and if they understand how they should proceed with each step of our module. The choice to use a multiple choice format is due to time constraints as more conceptual fill in the blank tests will take too much time to test each individual and will also likely deter potential test takers due to how long it takes. Any simpler tests, such as true or false, will provide surface level information for our protocol as guessing is easily a factor that must be taken into account in a yes or no scenario. We will use t-tests to determine if there is significant difference in survey results between BTEC and ABET students. This result will allow us to determine if more background information is needed to make the protocol simpler to understand, as the ABET students have not previously studied the background of the lab module. The data gathered from our test will be organized into a spreadsheet and chart which will display the number of correct answers to our test on a graph and this number will provide us with information on how clear our protocol is to our readers and if the understanding of the material is clear and consistent.

The statistical data will be arranged as in the above graph and the number of correct answers will correspond to how well our protocol does in terms of clarity, while the sheer number of tests in a certain category will allow us to more accurate say that for example, 22 test takers were able to achieve a 9 out of 10 on our statistical test so our protocol is interpreted as planned and will be easily reproducible. The t-tests between the ABET and BTEC students will allow us to determine statistical differences between the two groups and this will allow us to determine is there is a large difference or if there is none. The results of testing will allow modifications to the protocol to improve the overall understanding of the process and background, as well as the ability to reciprocate the protocol. Since the current testing plan does not involve carrying out the lab procedure, the future of the design can include testing the actual procedure and establishing baseline values. Once all of the procedures and results can be consistently monitored, the module can be implemented as an alternative in order to look at the extent of differentiation by quantifying calcium in the osteoblasts. Ethical considerations for our project include obtaining the necessary osteoblast cell lines for testing and generally the cell lines obtained will be from animal sources so there will always be the issue of how to humanely obtain these cells. Especially in the case of human osteoblast cell lines, there will be specific procedures which will have to be followed and any testing done on these cells will reflect personal information of that individual so proper disclosure of results and genetic information should be disclosed according to current standards. Trypsin: Hazardous in case of contact with eye or inhalation. Slight irritant of the skin. No information available on carcinogenicity, mutagenicity, teratogenicity, and developmental toxicity. PBS: Low hazard for handling. May cause skin, eye, and digestive or repertory tract information. Fetal Bovine Serum: No toxicity from inhalation. Mildly irritating to mouth. May cause skin or eye irritation. Paraformaldehyde: Very hazardous in case of skin and eye contact. Eye contact can lead to blindness, skin contact can lead to inflammation and blistering. Inhalation can lead to burning,

sneezing and coughing. Prolonged inhalation can lead to choking, unconsciousness and even death. No information available on carcinogenicity, mutagenicity, teratogenicity, and developmental toxicity. Tris Buffer: Causes eye and skin irritation. Causes severe gastrointestinal tract irritation, live and kidney damage. May cause hypoglycemia and hyperkalemia. May cause irritation of the respiratory tract. Calcium Assay Buffer: Causes skin, respiratory, and eye irritation. Also irritates if ingested or injected.

MSDS Citations Trypsin; MSDS; Science Lab: Houston, Texas, http://www.sciencelab.com/msds.php?msdsId=9927313 Phosphate Buffered Saline; MSDS; Fisher Scientific: Fair Lawn, New Jersey, http://tinyurl.com/lo8rj9x Fetal Bovine Serum; MSDS; Sigma: St Louis, Missouri, http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en& productNumber=F2442&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldric h.com%2Fcatalog%2Fproduct%2Fsigma%2Ff2442%3Flang%3Den Paraformaldehyde; MSDS; Sigma, St Louis, Missouri, http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en& productNumber=P6148&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.c

om%2Fcatalog%2Fproduct%2Fsial%2Fp6148%3Flang%3Den Tris Buffer; Science Lab, Houston Texas, http://www.sciencelab.com/msds.php?msdsId=9927020 Calcium Assay Buffer (10x); Cayman Chemical Company, Ann Arbor, Michigan, https://www.caymanchem.com/msdss/700550m.pdf