Please find below examples of visualization of cells in QGel™ MT 3D Matrices using fluorescence microscopy:

1. download PDF Protocol for investigating cell viability

2. download PDF Protocol for staining of nuclei and F-actin filaments.

 

 

1. Protocol for investigating cell viability (Live/Dead assay)

ABOUT THIS PROTOCOL: This is an example of Live/Dead assay protocol that can be also found in published literature (e.g. Reaber, Biophysical Journal 2005). Note that the protocol below is only a guideline. Volumes and concentrations of the different chemicals and incubation times require to be optimized for your specific experimental conditions.

 

 

Required chemicals/solutions for this assay:

• LIVE/DEAD® Viability/Cytotoxicity Kit, for mammalian cells (from Molecular Probes™, Invitrogen detection technologies). This kit contains two vials of Calcein AM (4mM) and two vials of Ethidium homodimer-1 (2mM)

 

Prepare the following solutions:

• Solution A: 1:40 ratio of Calcein AM and 1:16 Ethidium homodimer-1 in PBS solution
• Solution B:  pre-warmed culture media (37°C)

 

1. Transfer desired gel samples with encapsulated live cells in a new well plate (under cell culture hood) and wash them once with pre-warmed PBS (i.e. 37°C) at room temperature (RT) for 5-10 min
2. Dilute solution A at 1:50 ratio in solution B and add this staining solution to the well with the gel that was previously washed
3. Incubate 30-45 min in cell culture incubator (37°C)
4. 1x quick wash with PBS. Protect from light! 
5. Store the gel in PBS and image the sample as soon as possible. Protect from light! 

Important note 1: For efficient staining always make sure that gels with the encapsulated cells are submerged in the different solutions and not floating on the solution surface.

 

Important note 2: If you have a large number of samples to image, it is recommended to split the experiment with no more than 2-3 samples to visualize at each time. Best results are achieved with freshly stained samples (max. 30-45 minutes old, before being imagined). 

 

 

2. Protocol for staining of nuclei and F-actin filaments

ABOUT THIS PROTOCOL: This is an example of staining protocol that can be found also in published literature (e.g. Reaber, Biophysical Journal 2005). Note that this protocol below is only a guideline. Volumes and concentrations of the different chemicals and incubation times require to be optimized for your specific experimental conditions.

 

 

Prepare the following solutions:

• 0.1M Glycine solution in PBS
• Fixation solution/permeabilization: 3-5% Paraformaldehyde (handling precautions are required as it is very toxic) + 0.2% Triton-X100 in PBS at 37°C.

Note: prepare the triton solution separately and add it to the paraformaldehyde solution only at time of fixation.

• 1% w/v BSA solution in PBS
• F-actin filament staining solution: approx. 0.8U/mL (Rhodamine or ALEXA conjugated Phalloidin) in 1% BSA solution in PBS.

Note: You must also follow instructions provided by the manufacturer of the different stains. Rhodamine or ALEXA conjugated Phalloidin are normally stored at higher concentrations and are diluted to working concentrations immediately before use. Concentration may be varied depending on experimental conditions.

(caution: they are normally provided as powder which required particular handling precautions, as they are very toxic)  

• Nucleus staining solution: For example, prepare a 2.5ug/mL solution for DAPI

Note: You must also follow instructions provided by the manufacturer of the different stains. Stains are normally stored at higher concentrations and are diluted to working concentrations immediately before use. Other nucleus stains, such as PI, DRAQ5, etc. can also be used instead of DAPI.

 

A. Fixation and permeabilization: 

 

1. Transfer desired samples in a new well plate (under cell culture hood) and wash them once with PBS at room temperature (RT) for 10 min
2. Add the fixation/permeabilization solution to each gel sample
3. Incubate at RT for 30-50 min (on shaker is preferable but not necessary)
4. 1x wash with 0.1M Glycine at RT for 15-30 min
5. 2x washes with PBS at RT for 5-10 min

 

Important note: For efficient fixation always make sure that gels with the encapsulated cells are submerged in the different solutions and not floating on the surface.

 

B. Actin filament staining: 

(e.g. with Rhodamine-conjugated Phalloidin).

 

1. Add the F-actin filament staining solution, and incubate at RT for 1-2h (on shaker is preferable but not necessary). Protect from light!
2. 2x washes with PBS at RT for 5-10 min (on shaker is preferable but not necessary). Protect from light!

 

Important note: For efficient staining always make sure that gels with the encapsulated cells are submerged in the different solutions and not floating on the surface.

 

C. Cell nuclei staining: 

(e.g. with DAPI, DRAQ5, PI, etc.)

 

1. Add the nucleus staining solution and incubated at RT for 40-50 min (on shaker is preferable but not necessary). Protect from light!
2. 2x washes PBS at RT for 5-10 min (on shaker is preferable but not necessary). Protect from light!
3. Optional: samples can be mounted in conventional mounting media for longer staining conservation.

 

Important note: For efficient staining always make sure that gels with the encapsulated cells are submerged in the different solutions and not floating on the surface.