Apoptosis in HEK cells: Protein Extraction and Microscopic Observations Assessments and Preparation related this lab: • Complete the Pre-lab Quiz online before coming to lab. (Remember that this quiz will also include questions relating to the lab that yo

Rate this post

.

Apoptosis in HEK cells: Protein Extraction and Microscopic Observations Assessments and Preparation related to this lab: • Complete the Pre-lab Quiz online before coming to lab. (Remember that this quiz will also include questions relating to the lab that you did last time!) • Bring in a hard copy of the Introduction (including a References Cited section). • Do the BSA dilution calculations on page 7 (Table 1). Your instructor or TA will check your values. • Bring along Lab 1 as a reference for microscopy and pipetting. • Write a Results section based on today’s work to pass in at the beginning of Lab 3. If we knew what we were doing, they wouldn’t call it research. ~Albert Einstein Last week you learned how to grow and maintain mammalian cells in culture. This week you will use these same cells for an apoptosis experiment. Pairs of students will be assigned to prepare either treated or control cells. Concentrated hydrogen peroxide (H2O2), which will cause oxidative stress to the cells, will be added to the culture media of the treated cells, and distilled water will be added to the culture media of the control cells. After letting the cells incubate for 30 minutes, you will work with them in two different ways: 1) Cells will be examined microscopically. Apoptotic cells progress through a series of defined morphological changes. You will make two types of observations: a. Trypan Blue viability test (just like what you did last lab!) b. Brightfield or phase contrast to look at morphology (i.e. blebbing, cell size/shape) 2) Cells will be lysed and the protein extracted. The protein concentration in this cell lysate will be quantified via a Bradford Assay. The cell lysate will be diluted and boiled with sample buffer, and then stored in the freezer for electrophoresis in Lab 3. As you discovered in Lab 1, it is labour intensive (and expensive!) to grow cells. Therefore, we will increase our efficiency by assigning pairs to prepare either the control cells or the treated cells and then the sample will eventually be shared with the other group. Note: there will be a lot going on in this lab, so it is important that you stay organized and carefully keep track of your photos and tubes of protein! Clear labelling is key ☺. 2 Division of Labour: “a” Groups (by the wall with the posters!) • Treat cells with H2O2 and extract protein (to determine protein concentration for Lab 3) • Take pictures of morphological observations for both Treated and Control cells “b” Groups (by the wall with the door!) • Treat cells with distilled water and extract protein (to determine protein concentration for Lab 3) • Viability test for both Treated cells and Control cells. PART A – Protocol to Induce Apoptosis: • Each pair will be provided with a round dish of HEK 293 cells (60 mm diameter). Remember that the cells are adherent and are growing on the bottom of the plate. • “a” groups will add 43.5 µl of 3% H2O2 to their dish (i.e. Treated cells). Give the dish a gentle swirl. Label the top of the dish with your group number (write directly on the lid), and place the dish on the provided tray. Your instructor or TA will place the tray into the CO2 incubator for 30 min. • “b” groups will add 43.5 µl of distilled water to their dish (i.e. Control cells). Give the dish a gentle swirl. Label the top of the dish with your group number (write directly on the lid), and place the dish on the provided tray. Your instructor or TA will place the tray into the CO2 incubator for 30 min. While you are waiting, you should: 1) Carefully read Part B. Locate 5 tubes (1 Falcon tube; 4 microcentrifuge tubes) that you’ll need for Part B; write your group # directly on them with Sharpie. Add 50 µl of trypan blue to a microcentrifuge tube. 2) Go to page 7. Label your cuvettes (which look like glass test tubes) using small pieces of tape near the top) and add the correct amount of PIPES buffer to cuvettes 1-10 (use Tables 1 and 2). Whiteboard at front Wall with the posters Group 1a Treated cells – protein extraction & take microscopy pictures Group 1b Control cells – protein extraction & viability test Wall with the door Group 2a Treated cells – protein extraction & take microscopy pictures Group 2b Control cells – protein extraction & viability test Group 3a Treated cells – protein extraction & take microscopy pictures Group 3b Control cells – protein extraction & viability test Group 4a Treated cells – protein extraction & take microscopy pictures Group 4b Control cells – protein extraction & viability test Group 5a Treated cells – protein extraction & take microscopy pictures Group 5b Control cells – protein extraction & viability test Group 6a Treated cells – protein extraction & take microscopy pictures Group 6b Control cells – protein extraction & viability test Windows at back 3 PART B – Protocol for Extracting Protein from Cells: (all groups do this!) 1. Label a 15-ml Falcon tube (group # and either “treated” or “control”). After the 30 minute incubation, retrieve your dish of cells. 2. Use a P1000 to collect the media and transfer it into your labeled Falcon tube. Remember: press the micropipettor plunger to the first stop before putting the tip in the liquid to reduce air bubbles. 3. Use a P1000 to gently add 900 µl of PBS onto the cells in the dish. Using a fresh tip, add another 900 µl of PBS onto your cells, so you have a total of 1.8 ml in the dish in total. Give your dish a swirl and immediately use the same pipette to transfer the PBS into your labeled Falcon tube. 4. Use a P200 to add 200 µl of trypsin onto the cells that may be remaining in the dish. As we saw in Lab 1, this will dissociate the cells from the bottom of the plate. This process may take a few minutes. Make sure to rotate the plate from side to side to ensure that the cells have lifted. 5. Use a P1000 to add 900 µl of DMEM media onto the cells + trypsin in the dish. Pipette up and down gently. You should be able to see that the cells have lifted. Note that the Control cells might take a little more time to lift. Tap the sides of the dish to see if that helps. Transfer the media (containing your cells) into the labeled Falcon tube. Repeat with another 900 µl of fresh DMEM media. These steps ensure that all the cells have now been transferred to the Falcon tube. 6. Bring your Falcon tube to the large centrifuge on the side bench. Balance it with another group’s tube and spin for two minutes. Your TA or Instructor will help with this. 7. After centrifuging, you should see your cell pellet in the bottom of the Falcon tube. Carefully pour off the supernatant into the glass waste beaker. 8. Using a P1000, add 1800 µl (so 900 µl twice) of PBS to the cell pellet. Gently pipette the cells (now in PBS) up and down to resuspend the pellet. 9. Label a microcentrifuge tube (group # and either “Treated” or “Control”). Using a P200, transfer 50 µl of the resuspended cells into the microcentrifuge tube and add 50 µl of trypan blue to it. This tube will be shared with the other group on your bench. You will use the contents of this tube for the cell viability test and morphology observations. 10. Spin the Falcon tube (containing the resuspended cells in PBS) again for 2 minutes in the large centrifuge on the side bench. 11. Using a P1000, carefully remove the supernatant (ie. the PBS) and dispose of it in the waste beaker. Be careful to not suck up the cell pellet. Place the Falcon tube (containing the cell pellet) on ice (~1 minute). 12. Label a new microcentrifuge tube and place it on ice. Use a P200 to add 200 µl of cold RIPA buffer (it’s in your ice bucket) to your cell pellet in the Falcon tube and resuspend the cells. Immediately transfer everything to your labeled 1.5 ml microcentrifuge tube on ice. Set a timer for 12 minutes