Genomic and Regenerative Medicine Discussion Paper

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Genomic and Regenerative Medicine Discussion Paper

7029BMS Lab Schedule 

Analysis of the effects of THP-1 derived exosomes on HepG2 cells

 

Exosomes are microscale extracellular vesicles that are present in most somatic fluids including blood and urine, as well as the media of cell cultures. These-lipid-bearing microvesicles range in size from 30 to 200 nm. They often contain key biomolecules like proteins and nucleic acids (both DNA and RNA) derived from their cell of origin. Recent studies support that exosomes are involved with intercellular communication, including between macrophages and cancer cells. E.g. https://pubs.rsc.org/en/content/articlelanding/2020/ra/c9ra07332a#!divAbstract

and

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290460/

and

https://www.cell.com/molecular-cell/fulltext/S1097-2765(10)00451-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS109727651000451X%3Fshowall%3Dtrue#secd5383008e932

Exosomes therefore likely have important roles in health and various diseases.

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Below is a set of procedures that have been put together to investigate if exosome-mediated communication between macrophages and liver cancer cells can induce changes in cell phenotypes/behaviour. In general, the instructions will be less detailed than those you followed in 7027BMS, as you are expected to take charge of your own preparation for lab sessions. You will need to read the required information before each session as well as seek additional information (and watch online videos if needed) to ensure you understand what you need to do before coming into the lab. The 7029BMS labs are designed to provide you with an experience similar to that you would encounter in a research lab, where you are expected to be independent and able to plan experiments yourself, often by adapting published procedures to your needs.

 

 

 

Session 1

 

Purification of exosomes

(based on the protocol develop by https://www.tandfonline.com/doi/full/10.3402/jev.v3.23430)

 

Work in a group of 3

  1. Collect a flask of THP-1 cells – this a non-adherent cell line that produces exosomes. These cells were cultured in a 5 ml culture flask in normal media (RPMI 1640 + 2 mM Glutamine + 10% Foetal Bovine Serum) before being treated with 0.5 nM phorbol myristate acetate (PMA) for 3 h and then placed by back in normal media for a further 24 h.
  2. Mix the cells and culture media in the flask gently and transfer to a clean centrifuge tube, spin the tube (with a suitable balance) for 5 minutes at 2000rpm to pellet the cells from the supernatant.
  3. Gently pour off the supernatant to a new tube and dispose of the cell pellet in the appropriate discard container. Place your tube with the supernatant on ice.
  4. Take the provided 10 ml of sepharose solution (sepharose CL-2B washed with PBS-citrate 0.32% (w/v) pH 7.4, 0.22 µm filtered) and gently swirl to resuspend. Gently pour the solution steadily into the provided syringe (capped at the end using parafilm) and allow to settle. NOTE – step 4 may have been done for you.
  5. As described in the publication above, load 1.5 ml of your supernatant onto the column, and elute with PBS-citrate 0.32% (w/v) immediately collecting fractions of a suitable amount (see paper link above to find this information) in eppendorf tubes. Make sure all tubes are labelled with fraction number. Keep the fractions on ice.
  6. Add around 5 ml more PBS-citrate 0.32% (w/v) to the column to wash it and let the flow through drip into a waste beaker. You can move to the next step while this drips through. Once the dripping has stopped, cap the end and add more PBS-citrate 0.32% (w/v) to prevent the column from drying out.
  7. Pick 3 fractions you think will contain exosomes (according to the paper above).
  8. Each member of the group will then pick and work with one of the 3 fractions from now on. Make sure your fraction tube is labelled with your name and fraction number.
  9. Transfer 0.25 ml of your fraction to another Eppendorf tube. This will be used for RNA extraction. Make sure this tube is also labelled with your name and fraction number and RNA.
  10. Give your 2 tubes to the academic who will store them at -80ºC until required.

 

 

 

 

 

Session 2

Each student within the group from session 1 should process one of the fraction samples

 

PKH67 staining of THP-1 derived exosomes

  1. You will have a tube on your desk containing 1.5 µl of the dye PKH67. Add 0.2 ml of DILUENT C to this tube and mix the tube gently.
  2. Add 200 µl of the PKH67/DILUENT C mix to 200 µl of the fraction supernatant you identified that you think will contain exosomes and gently mix continually for 30 seconds using a 1 ml pipette (and blue tip). Incubate your tube at room temperature for 5 minutes.
  3. Quench by adding 0.4 ml 1% BSA in PBS. Incubate your tube at room temperature for 5 minutes.

 

Incubation of HepG2 cells with THP-1 exosomes for flow cytometry analysis

  1. Using the Cat 2 cabinets, add 0.4 ml of your labelled fraction to a flask of HepG2 cells. Control flasks where nothing is added will be analysed alongside your test samples on (i.e. you do not need to pick up a control flask). Clearly label your flask.
  2. Incubated at 37ºC until a later session where they will be analysed by flow cytometry

 

Incubation of HepG2 cells with THP-1 exosomes for scratch wound healing assay

  1. Using the Cat 2 cabinets, add the remaining 0.4 ml of your labelled fraction to a well within a 6-well plate of HepG2 cells. A 2nd well will act as a control. Clearly label the wells. The other 4 wells will be used by other students (i.e. there will be 3 students per 6-well plate).
  2. Using a sterile 100 µl pipette tip (usually yellow), scratch a straight line down the middle of each well. Make sure to use a different pipette tip for each sample.
  3. Have a look at your plate under the microscope and take an image of each scratch (Time 0 timepoint). An academic will be at the microscope to show you how to do this.
  4. The plates will then be incubated 37ºC and images will be taken for you over the next 48hr.

 

 

 

 

 

Session 3

 

You will use the TaqMan MicroRNA Assay to assess the levels of miR-150 in your chosen exosome sample. Read information from below links:

TaqMan Small RNA Assays Quick Reference Card:

http://tools.thermofisher.com/content/sfs/manuals/cms_083619.pdf

 

Further information:

https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fbrochures%2Fcms_078025.pdf&title=QXJ0aWNsZXMgJmFtcDsgV2hpdGUgUGFwZXJzOiBEZXNpZ24gUGlwZWxpbmUgZm9yIFRhcU1hbiZyZWc7IFNtYWxsIFJOQSBBc3NheXMgKFRlY2hOb3RlcyBhcnRpY2xlKQ==

 

General guide:

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/4364031_TaqSmallRNA_UG.pdf

 

TaqMan™ MicroRNA Reverse Transcription Kit:

https://www.thermofisher.com/order/catalog/product/4366596#/4366596

 

TaqMan® Universal PCR Master Mix II, no UNG:

https://www.thermofisher.com/order/catalog/product/4440043#/4440043

 

TaqMan™ MicroRNA Assay:

https://www.thermofisher.com/order/catalog/product/4427975#/4427975

 

miR-150 specific RT primer and PCR probe and 2 primers:

https://www.thermofisher.com/order/genome-database/details/microrna/000473?CID=&ICID=&subtype=

 

U6 specific RT primer and PCR probe and 2 primers:

https://www.thermofisher.com/order/genome-database/details/microrna/001093?CID=&ICID=&subtype=

 

ThermoFisher Real-Time PCR (qPCR) Learning Center:

https://www.thermofisher.com/uk/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center.html

 

RNA extraction

  1. Add 0.5 ml of TRIzol reagent to the 0.25ml of fraction you put aside for RNA extraction in week 1.
  2. Incubate sample for 5 minutes at room temperature. Add 0.2 mL of chloroform per 1 mL of TRIzol used. Cap tube securely and shake vigorously by hand for 15 seconds.
  3. Incubate sample for 3 minutes at room temperature. Centrifuge sample at 13,000 RPM for 15 minutes at room temperature. Remember to have the centrifuge balanced (possibly with other peoples sample(s)). The sample will separate into a pale green, organic phase, an interphase, and a colourless upper aqueous phase.
  4. Transfer very carefully to another tube the phase that contains the RNA (you must find this information yourself if you are unsure which layer contains the RNA). Precipitate the RNA by mixing with cold isopropyl alcohol. Use 0.5 mL of isopropyl alcohol per 1 mL of TRIzol used. Incubate samples for 10 minutes at room temperature then centrifuge at 13,000 RPM for 10 minutes. Remember to have the centrifuge balanced.
  5. Remove the supernatant and wash the pellet once with 75% ethanol, adding 1 mL of ethanol per 1 mL of TRIzol used. Vortex samples and centrifuge at 13,000 RPM for 5 minutes. Remember to have the centrifuge balanced.
  6. Remove all ethanol, air-dry for 5 minutes, and then dissolve in 20 µl PCR water or DEPC-treated water by pipetting the solution up and down. Incubate for 10 minutes at 60°C if necessary.
  7. Quantify your RNA using the nanodrop and record your values (you will need to know the concentration when preparing your reverse transcription reactions)
  8. Ensure your tube is labelled with you name, fraction number and RNA.
  9. Keep samples on ice.

 

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Reverse Transcription (RT)

  1. You will be given 15 μl of the master mix. Before the session, read the RT instructions from the TaqMan Small RNA Assays Quick Reference Card to ensure you understand the contents of the master mix that has been prepared for you.
  2. Add 7 μl of the master mix to 2 PCR tubes (smaller than eppendorfs).
  3. Follow the rest of the TaqMan Small RNA Assays Quick Reference Card protocol from step 3 (note- you are preparing single-stranded RNA, so follow d.). In terms of which RT primer to add to each tube: 2 tubes should be split as follows: Tube 1- RNA prep + miR-150 RT Primer; Tube 2- RNA prep + U6 snRNA RT Primer. Before the session, you will need to use your Nanodrop data to determine how much you have to dilute your RNA prep by (using DEPC-treated water) to have 1-10 ng total RNA in the reaction.
  4. Place your clearly labelled PCR tubes (smaller than Eppendorf tubes) in the ice bucket next to the thermal cycler.
  5. The thermal cycle will perform a run using the following conditions (will take about an hour):
  • Mode: Standard
  • Reaction volume: 15 µL
  • Thermal cycling conditions:

Step Time Temperature

Hold 30 minutes 16°C

Hold 30 minutes 42°C

Hold 5 minutes 85°C

Hold ∞ 4°C

Session 4

 

Flow cytometry analysis of HepG2 cells

  1. Using the Cat 2 cabinet, remove media from the plate and add to 15 ml falcon tube.
  2. Add 3 ml PBS to plate to wash cells, remove PBS and add to the media in the 15 ml falcon tube.
  3. Add 0.5 ml trypsin to plate and put to 37°C for 5 minutes
  4. Add 3 ml PBS, mix using the pipette and add cells/PBS to the 15 ml falcon tube
  5. Spin 1,000 RPM for 5 minutes, pour off supernatant, add 5 ml PBS and re-suspend cell pellet, then spin again 1,000 RPM for 5 minutes
  6. Pour off supernatant, add 5 ml PBS and re-suspend cell pellet, then spin again 1,000 RPM for 5 minutes
  7. Resuspend after your final spin in 1 ml PBS and place sample in a labelled Eppendorf tube.
  8. Take your sample to the BD Accuri flow cytometer and run your samples using count and FITC as your experimental axis, run 10 000 cells through for each run.
  9. Control samples (no exosomes added) will similarly be analysed.
  10. Save your data to a memory stick.

 

 

 

Quantitative PCR (qPCR) amplification of RT reaction samples

Before the session, read the Quantitative PCR (qPCR) amplification instructions from the TaqMan Small RNA Assays Quick Reference Card to see how to prepare required 4 tubes containing the reagents below:

  • TaqMan® Universal PCR Master Mix II, no UNG
  • Nuclease-free Water
  • TaqMan Small RNA Assay (20X) (1 probe and 2 primers for miR-150 OR 1 probe and 2 primers for U6 snRNA)
  • Product from RT reaction (one of the 4 tubes)

 

4 tubes will be set up for you containing 18.67 μl of a master mix containing all of the reagents except your product from the RT reaction:

  • Tube 1 miR-150 RT reaction with miR-150 Taqman Small RNA Assay – duplicate 1
  • Tube 2 miR-150 RT reaction with miR-150 Taqman Small RNA Assay – duplicate 2
  • Tube 3 U6 snRNA RT reaction with U6 snRNA Taqman Small RNA Assay – duplicate 1
  • Tube 4 U6 snRNA RT reaction with U6 snRNA Taqman Small RNA Assay – duplicate 2

 

  1. Follow the published instructions to add the correct RT reaction to each tube in the correct amount.
  2. Take the samples to an academic who will add them to a qPCR plate. Note- no template controls (i.e. water added instead of RT reaction) will also be run on the plate for both the miR-150 Taqman Small RNA Assay and U6 snRNA Taqman Small RNA Assay reactions.
  3. The qPCR will then be run as instructed in the “Set up and run the real–time PCR instrument” section of the general guide.
  4. Results will be uploaded onto Aula.