Tag Archives: protocols

Laboratory, Project

H & E Staining – Protocol Reminder

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Today, I plan to stain the cut glass, glass vessels and T25 Flasks.

Before I head over to the lab, I always review the protocol and make sure I have an easily accessible copy.  While it is simple, I have not done it often enough to remember the process without error.

BASIC H&E STAIN:

  1. Remove PBS
  2. Add Hematoxylin – leave for 5 min
  3. Rinse under running water
  4. Add Ammoniated water for 30sec (2 – 3 drops ammonia  to 400mL distilled water)
  5. Rinse under running water
  6. Add Eosin for 2 min
  7. Rinse under running water
  8. Add 95% Etoh for 30sec with agitation
  9. Add 100% Etoh wash x 3

I still need to check with the lab manager if I they are happy that I preserve the stained cells in resin for removal from the lab.

Reviewing the protocol also ensures that I check materials prior to starting the process – there is nothing worse than starting a protocol only to discover that some of the materials are missing.

Creative Development, Laboratory, Project

Immunostaining Protocol

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It is time to complete the immunostaining protocol with guidance from Jo-Maree. I must admit that with holidays looming, my note taking was a bit sketchy. I will need to follow up with Jo-Maree to record the correct details of DAPI and Fluorescein Phalloidin stain. This will ensure that I know how to prepare (and order) stocks in the future.

I already prepared a couple of wells at different cell concentrations ready for staining.  We had to delay the protocol, so some of the higher concentration wells are likely a bit over-confluent. It will be interesting to see how they look under the fluorescence microscope.

CELL FIXATION: ‘Dirty’ Biolab

Working in Fume HoodWorking with 4% PFA in Fume Hood

Prior to imaging, I fixed the cells in 4% PFA:

  1. Remove culture media (discard in waste container with bleach)
  2. Wash cells with PBS (discard in waste container with bleach) x 2
  3. Move cells to fume hood
  4. Add 4% PFA to each well for 15 – 20min at room temp (in fume hood)
  5. Remove 4% PFA solution (discard in PFA waste container in fume hood)
  6. Add PBS (make sure cells are covered or they dry out and produce poor images

IMMUNOSTAINING: At lab bench area

Lab BenchWorking at lab bench in the Stroke Group area

  1. Remove PBS from each well
  2. Add 1mL 0.3% Triton X-100 (a strong detergent) to permeabilize cells (make cells permeable – this allows the phalloidin stain to enter the cell structure) for 10 min
  3. Make up DAPI (5mL PBS Tween and 1ul DAPI) and protect from light with aluminium cover
  4. Remove Triton X-100 and add 1mL DAPI solution to each well and incubate at room temp (with aluminum cover to protect from light) for 5 minutes
    Aluminium Cover
  5. Make up Flouroscein Phalloidin (1mL PBS sand 2μL Flouroscein) and protect from light
  6. Remove and discard DAPI solution
  7. Add 1mL PBS 0.1% Tween to each well for 5 min then discard x 3 (i.e. wash with PBS Tween x 3)
    PBS Tween
  8. Add Phalloidin stain to each well.
  9. Incubate at room temp for 1 – 2 hours
  10. Remove Phalloidin solution
  11. Wash with PBS Tween x 2
  12. Add 1mL PBS in each well
  13. Cover with aluminum foil to protect from light.
  14. Cells are ready for imaging. They can be stored in the fridge (with aluminium foil cover) until ready.

 

Creative Development, Laboratory

Immunostaining of fibroid cells

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To gain some more insight into the cellular structure of my fibroid cells, I asked Jo-Maree to help me with immunostaining. Immunostaining refers to a staining method that uses antibodies to stain different proteins and structures within the cell. We are going to start with an easy protocol using two antibodies: DAPI and Phalloidin.

DAPI (4′,6-diamidino-2-phenylindole) is a very common fluorescent blue stain used to reveal the nucleus in cultured cells. It can penetrate the intact membrane of the cell. Thus, it can be used for staining both fixed and live cells.

Microscopic image of stem cells, Hues 9 stained with DAPI (blue)Microscopic image of stem cells, Hues 9 stained with DAPI (blue) by the UC San Diego Stem Cell Program.

BASIC LIVE CELL STAINING PROTOCOL

  1. Add DAPI to the complete culture medium used for cultured cells at a concentration of 10 ug/mL.
  2. Remove culture medium from the cells and replace with the medium containing the DAPI.
  3. Cover cells from light exposure and incubate at room temperature or 37°C for 5-15 minutes, then image.

Direct Addition: According to the biotium protocol, you can also stain cells by adding the dye directly to the cell culture and medium. However, this requires a 10X concentration of dye. 

  1. Add the dye to complete culture medium at 10 times the final recommended staining DAPI concentration – 100 ug/mL..
  2. Without removing the medium from the cells, add 1/10 volume of 10X dye directly to the well.
  3. Immediate mix thoroughly by gently pipetting the medium up and down. For larger well sizes (e.g., 24-well to 6-well plates), the plate can be gently swirled to mix.
  4. Cover cells from light exposure and incubate cells at room temperature or 37°C for 5-15 minutes, then image

STAINING FIXED CELLS/TISSUES:

  1. Add DAPI to PBS at 1 ug/mL.
  2. Add the PBS with dye to cells or tissue sections and incubate at room temperature for at least 5 minutes with covering from light exposure.
  3. Samples can be stored in a lightsafe covering (e.g. aluminium foil) at 4°C after staining and before imaging

From: https://biotium.com/tech-tips/protocol-staining-cells-with-hoechst-or-dapi-nuclear-stains/ with minor edits for simpliticy.

Since we are using phalloidin and fixing the cells prior to imaging, we need to follow some additional step to make the cells permeable.

Phalloidin Jo-Maree did not have any relevant stocks, Natalie kindly sourced a sample from another group. [I am always impressed by the generosity of functioning labs and how groups are happy to share stocks to enable other researchers to move forward.]

We are using Fluorescein Phalloidin as a counterstain to enable the visualisation of actin – a protein found in large quantities in the cytoskeleton and cell muscle fibres. As such, it  plays a vital role in cell muscle contraction and overall cell movement.

Interestingly Phalloidin is a toxin (specifically phallotoxin) derived from Amanita phalloides (death cap mushroom).

Amanita phalloidesImage of Amanita phalloides via Wikimedia Commons. 

Phalloidin binds very well to actin filaments and is therefore very useful in visualising cell structure.

Phalloidin staining of actin filamentsU2OS cells stained with fluorescent phalloidin taken on a confocal microscope by Howard Vindin

STAINING FIXED CELLS

  1. Fix cells in 3–4% formaldehyde in PBS at room temperature for 10–30 minutes.
  2. Remove fixation solution and wash cells 2–3 times in PBS.
  3. Add 0.1% Triton X-100 in PBS into the fixed cells for 3–5 minutes to increase permeability. Then wash cells 2–3 times in PBS.
  4. Add phalloidin-conjugate working solution. Incubate at room temperature for 20–90 minutes.
  5. Add DAPI DNA staining dye at this point.
  6. Rinse cells 2–3 times with PBS, 5 min per wash.
  7. Cover with lightfast material to preserve fluorescence

Adapted from:  https://www.abcam.com/protocols/phalloidin-staining-protocol 

Creative Development, Laboratory, Project

Cut Glass Collection

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As part of the residency project, I have started a collection of cut glass items. These were sourced from different second hand shops and build on an existing collection of items used for an exhibition at The  Edge at the State Library of Queensland in 2013.

I am particularly attracted to the patterns of the glass. A recurring central motif in many items is a star.

Cut Glass DishClear cut glass dish – approx 12cm diameter with central star motif and radiating pattern.

This links to my current interest in deep time including the birth of the universe and emergence of complexity. The glass items also look wonderful when lit from rear.  As such, I am considering mounting them over a light source. However, this remains to be seen…

In order to grow cells in the dishes, they need to be sterilised so that they do not carry any bacteria or other organisms that could contaminate my cells.

I am feeling more confident in using the benchtop autoclaves independently so am preparing a batch for sterilisation today. As per previous work, they are placed in autoclave bags and sealed with tape. Once the bags are autoclaved, black lines indicate successful sterilisation.

Cut Glass DishCut glass dish and wrapped dish ready for sterilisation.

I have also sources some small glass vials which I am considering integrating into some of the future creative works. There are various shapes that I am planning to test.

Glass VialsSelection of glass vials for cell culture trial including metal closures. 

Collection of Glass Items in Autoclave Bags Glass vessels in autoclave bags ready for sterilisation. 

Finally, I have also prepared some additional 150mm and 90mm Petri dishes. The large dishes will be used as container vessels for the cut glass dishes to keep them sterile during cell culture.

Petri dishes and other glassware ready for autoclavingPetri dishes and other glass items in autoclave bags ready for sterilisation. 

I divided the batch into two runs. As per previous process, I used cycle 6 (134 degrees for 10 min). This enables me to process both glassware and metal.  It takes about 10 min for the sterilization process (but extra for cooling to handle materials).

Autoclave InstructionsAutoclave instructions with cycle details. 

Autoclaved dishesAutoclaved bags containing sterilised Petri dishes. 

Autoclaved items stored in labAutoclaved bags stored in lab area, ready for use.

Creative Development, Laboratory, Project

Hematoxylin and Eosin Staining

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Jo-Maree finally had some time to go over basic H&E staining procedures. Since my HBVPs are fixed on the base of  glass Petri Dishes, the process is much less involved than working with wax embedded specimens.

H&E is a very common stain combination used in histology. Hematoxylin stains nuclei blue-purple
Eosin stains cytoplasm (protein, muscle fibres etc.) pinkH & E Stain Protocol Basic H&E staining protocol from Jo-Maree.   We only need to follow the staining process.

Stain: washing Petri Dish on bench in Histology Lab at MSP with Erlenmeyer flask containing distilled water for washing. 

Prior to adding the Hematoxylin stain, we washed the Petri dishes with distilled water (DW). Usually, we would simply wash the dishes under running water from the tap. However, since rapid water could dislodge the cells from the base of the dish, we have used a beaker to control the water flow.  I washed each dish twice to remove PBS and dislodged cells.

Hematoxylin StainHematoxylin Stain – deep red stain 

Contrary to what the name Hematoxylin suggests, the dye is actually naturally derived and comes from the tree  Haematoxylum campechianum (Logwood). As such, it is non-toxic and does not need to be added in a fume cabinet. The dye was added to the Petri Dishes for 5 mins, then washed with distilled water.

The next step involved adding ammoniated water (approx 2 – 3 drops ammonia to 400mL distilled water) to the stained cells for 30 secs.   This process is referred to as ‘bluing’ and helps change the red – purple hematoxylin to a blue – purple color.

Hematoxylin Stained DishCells visible on the base of Petri Dish following Hematoxylin staining.

After washing the Petri Dish thoroughly after ‘bluing’, we added the Eosin stain.  Eosin is a xanthene dye and has an intense fluorescent colour.

Eosin StainEosin stain in Petri Dish.

The Eosin stain only needs 2 mins to stain the cytoplasm and matrix of cells. Following  another thorough wash of the dish, we added 95% ethanol and secured the Petri dish lids with parafilm.

For stained sections on glass slides, it is usual to add Xylene (toxic) and a coverslip. In this case, we could either create large scale glass covers (a bit impractical) or clear resin. I think clear resin is the best solution as it would create a barrier and preserve the dyed cells. I am keen to use the fixed cells in dishes as part of sculptural works.  However, I will need to check with lab manager David Steele that I am able to remove these fixed cells from the lab.

Laboratory, Project

Building a Stock of PHGL Tumour Baby Cells ….slowly

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My Tumour Baby cells still remain very sluggish and slow to replicate. I’ve been checking in regularly to chart their growth.

Tumour Baby Cells 17/09/21

Tumour Baby Cells 17/09/21

Brightfield microscope images of PHGL TB Cell Growth 17/09/21

As they have continued to grow, they have started to look less healthy and consistent. They remind me of gamma irradiated 3T3 cells (mouse fibroblasts that have been irradiated to stop replicating).  However, this could also be the result of using media that is not ideal, as we have been using some existing (expired) stock supplies of DMEM while we are waiting for our order to arrive.

Tumour Baby Cells 20/09/21

Tumour Baby Cells 20/09/21

Brightfield microscope images of PHGL TB Cell Growth 20/09/21

By 21/09/21 I decided there were enough cells to split into a second flask and freeze down one vial of cells. This will replenish the vial we used and bring our stock up to three vials.

Tumour Baby Cells 21/09/21

Tumour Baby Cells 21/09/21

Brightfield microscope images of PHGL TB Cell Growth 21/09/21

Since the Stroke team mainly work with HBVPs, I reviewed standard protocols for fibroblasts to determine an optimum freezer mix. The recommendation from a number of sources is to include a higher rate of FBS at 30%, 10% DMSO (anti-free agent) and 70% media with a min. of 1 x 106 cells. I made up a total of 1.5mL freeze mix (including cells).

When I passaged the cells, I added 1mL new media to the cells solution. Since the cells were not 80 – 90% confluent, I decided to split them at a rate of 2/3.  This means that the final freeze mix was: 150μL DMSO, 450μL FBS, 300μL media plus 600μL of cell mix. 1mL of this solution was added to cryovial and placed in a freeze box in the -80 degree freezer to be transferred into liquid nitrogen in the next day or so.

Since the cells were precious, I added the remaining cell freeze mix to a T25 flask with 5mL fresh media. There were also a few stubborn cells in the original T75 flask, (post passage),  so I added 10mL new media to see if any of them might grow.  Finally, the remaining 400μL cell mix (without freeze medium) to a new T75 flask with 10mL media.

At this point I had made up fresh DMEM media with the new batch of media, but decided to ‘wean’ the cells onto the new media at a 50/50 ratio of old to new. I am hoping that the new media will help the growth rate of the cells.

Creative Development, Laboratory, Project

LAB SCHEDULE

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UPDATED LAB SCHEDULE

Now that the project has the formal go-ahead, I am moving into lab mode and have determined some of the key milestones for the next months.

1: Training & Prep: 1 – 2 weeks

Training with HBVP cells include:

  • Thawing and culturing cells, making media, working in a biosafety cabinet and maintaining sterility, light microscopy
  • Learn to use the autoclave and prepare petri dishes and glass vessels for culture
  • Coat petri dishes and glass vessels with poly-l-lysine for cell adhesion, test with HBVP cells
  • Order media, reagents and kits
  • Submit IBC approval forms

2: Cell culture of fibroid cells – 4 – 8 weeks

  • Thawing and culture – grow up and freeze stocks of cells, light microscopy
  • Ask Dietmar to send 3D scaffolds
  • Grow and fix cells in petri dishes and glass vessels
  • Fluorescent microscopy of cells
  • Scanning Electron Microscopy (SEM) of cultured cells
  • Transmission Electron Microscopy (TEM) of cultured cells
  • Timelapse microscopy
  • 3D cell seeding HBVPs and Fibroid cells – see differences in cell response.
  • Wait for IBC approval

PROJECT: 3 months

3: Cell Immortalisation +

  • Immortalisation of primary fibroid cells via established commercial kit (Applied Biological Materials (ABM) or Alstem cell immortalisation kits)
  • Cell genetic profiling
  • Cell culture of immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Grow and fix cells in petri dishes and glass vessels
  • Timelapse microscopy

4: iPSC production

  • Reprogramming of primary cells to generate induced pluripotent stem cells (iPSCs) via established commercial kit (e.g. Epi5™ Episomal iPSC Reprogramming Kit available via Thermo Fisher)
  • Development of Gastruloids, Organoids or Neurospheres (self-organised 3D cell masses)
  • Cell culture of reprogrammed or immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Timelapse microscopy
  • If iPSC successful – create neurons and heart cells
Project

Ethics GRANTED! …but one more approval to go…

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We have approval to move forward with the key aims of the project. This is great news as it means I can start working with own cells. I am still a bit precious as there are limited vials, so I will do a couple of weeks of training on HBVPs before I move on to my own cells.

While we can get started on the fibroid cell culture, Brad realised that cell immortalisation will require further Institutional Biosafety Committee (IBC) approval. This is because the process will require the uses of lentiviral vectors. As such, it is considered Notifiable Low Risk Dealing (NLRD) and the committee will need to ensure that we have the appropriate facilities and training in place to move forward.  iPSC cell reprogramming is exempt, but we still need to let the IBC know what we are doing.

The application is due tomorrow, so we had a meeting this morning to go over the protocols and identify the particular kits we are going to use. There are a range of biomedical research supply companies, but the important thing is to make sure that we use an organisation that has an Australian supplier.  hTERT and SV40 T Antigen kits are the best options for our project as they are suitable for a range of  cell types including fibroblasts.  Fischer Scientific have Alstem Immortalisation Kits available, but ABM may also be a good option. They also have a good overview of Cell Immortalisation Protocols for anyone interested in the process.

For iPSC reprogramming, we are going to use the Epi5™ Episomal iPSC Reprogramming Kit by Thermo Fischer. Another group has used this product previously – so we can get tips on how to get the best results.  Lovely Jo-Maree is looking into the best purchasing options. With lead time for purchase and delivery, the products will likely arrive around the same time as final approval.

Project

iPSC Protocols

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We are still waiting for formal ethical clearance to undertake work using my fibroid (fibroblast) cells. In the interim, Brad sent through a Nature protocol detailing options for producing iPSCs from human keratinocytes derived from plucked hair follicles or skin biopsies. I have isolated keratinocytes from hair follicles before at QUT when I was part of the Tissue Repair and Regeneration Group for  the HSE (Human Skin Equivalent/Experience Project).  It was quite a mission as keratinocytes require a feeder layer of fibroblast cells. These needed to be irradiated to ensure that they did not outgrow the keratinocytes 🙁

Since we are only doing a skin biopsy if the fibroid cells are not viable, I am parking this option and scouting for protocols that are fibroblast specific. With that in mind, the company Sigma-Aldrich has specified an iPSC reprogramming protocol – Reprogramming of Human Fibroblasts using Non-Integrating Self-Replicating RNA Vectors – designed for fibroblasts and with a 30-day creation estimate. Of course, you always have to double or triple timeframes when you are undertaking a protocol for the first time. Of course, I will need to discuss this option with Brad and seek a pricing and availability estimate.

Fisher Scientific also has a reprogramming kit – the CytoTune-iPS Sendai Reprogramming kit. However, the latest version and full kit carries a hefty price tag at over $18, 500. They also offer a potentially more affordable option via ‘Episomal Vectors’ or Epi5™ Episomal iPSC Reprogramming Kit. These could be an option but Brad is of course the best advisor.

There are also a number of journal articles detailing iPSC reprogramming including: Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1 [Nature], Human Pluripotent Stem Cells (iPSC) Generation, Culture, and Differentiation to Lung Progenitor Cells [Methods Mol Biol.], Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells [Science Direct], Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system [Stem Cell Research & Therapy].

Looks like I’ve got some reading to do…

Project

Ethics in!

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Brad has sent through final information for Stage 2 ethics clearance.  We included a skin biopsy protocol in case the fibroid cells are unviable. Unfortunately, when dealing with primary cells, nothing is certain and there is a possibility that the initial cells are contaminated or do not recover from the freezing process. As such, we have included ‘Plan B’ to ensure we can move forward with the project and establish a new batch of primary cells for immortalisation.

The ethics documents were formally submitted through the Ethics Review Manager. Hopefully, I have addressed all project aspects and provided sufficient information for approval.  Fingers crossed!