Dil Labeling

Sugar Crush Detox

Natural Remedies for Food Cravings

Get Instant Access

This procedure is recommended for easy-to-handle NSC types growing as monolayers. A very simple procedure is to add DiI stock solution directly to the normal cell culture at a final concentration of 10 pg/mL. Cells should be at least 80% confluent; DiI may impair cell growth and survival. After incubation overnight under normal cell culture conditions, the cells are homogeneously and intensely labeled. The cells should be washed extensively prior to transplantation (at least 3X with PBS) to minimize transfer of dye to the host. It should be noted that DiI is transferred to host cells when transplanted cells die.

o kBLE 23.2 Cell labeling techniques - non-genetic labels

Technique

Common application

Advantages

Disadvantages

Lipophilic dyes, e.g. Dil

Amine reactive cell tracers, e.g. CFSE

BrdU labeling

Fluorescent nano-particles

Magnetic nanobeads/ small animal Magnetic Resonance Imaging

Short-term migration studies NSC - experimental brain tumor tracking studies

Short-term migration studies NSC - experimental brain tumor tracking studies

Additional ("back up") label

In vivo tracking of cells with multiphoton in vivo microscopy Macroscopic in vivo tracking of cells with in vivo imaging systems (e.g. IVIS 200)

In vivo tracking of cells with a small animal MRI device

Inexpensive Easy to implement No additional stains necessary after sectioning

Inexpensive Easy to implement No additional stains necessary after sectioning

Inexpensive Easy to implement Suitable for long-term studies if cells are not dividing

Easy to use

Relatively inexpensive

Suitable for in vivo tracking on a microscopic and macroscopic level Fewer animals necessary for time series experiments

Sensitive in vivo tracking of cells Fewer animals necessary for time series experiments Ferromagnetic beads can be easily detected in tissue section with Prussian Blue stains

Lipophilic dyes can leak out of transplanted cells In most cases only suitable for short-term studies (7-14 days) Dyes are being diluted when cells divide Additional staining is problematic

Dye might alter the cells' properties Dyes might leak out of transplanted cells Dyes are being diluted when cells divide In most cases only suitable for short-term studies

(7-14 days) Additional staining is problematic BrdU can be released by dying cells in vivo and taken up by phagocytic host cells BrdU is being diluted when cells divide Detection in sections needs harsh treatment which is not always compatible with co-stains Expensive equipment needed for in vivo studies Particles leak out of cells after tissue fixation

Very expensive, small animal MRI set-up necessary Technically challenging Nanoparticles might leak out of dead cells Only clusters of cells can be traced Several protocols using different Fe preparations are available kBLE 23.3 Cell labeling techniques - genetic labels

Technique

Common application

Advantages

Disadvantages

Viral transduction of cells with vectors encoding fluorescent proteins (FP)

Viral transduction of cells with vectors encoding LacZ

Viral transduction of cells with vectors encoding luciferase

Preparation of cells from FP-transgenic animals

Sex - mismatch (male cells in female animals)

Xenotransplantation, e.g. human cells in

Cell detection in tissue sections in vivo tracking of cells with multiphoton in vivo microscopy Tracking of cells with certain phenotype, i.e. Certain gene turned on if FP under specific promotor Cell detection in tissue sections Suitable for establishing transplantation procedures with quick feedback (see protocols in this chapter)

Macroscopic in vivo tracking of cells with in vivo imaging systems (e.g. IVIS 200)

In vivo tracking of cells with multiphoton in vivo microscopy Tracking of cells with certain phenotype, i.e. certain gene turned on if FP under specific promotor Additional ("back up") label Difficult transplantation studies, e.g. human cells into monkeys

Differentiation studies of human cells transplanted into rodents Additional ("back-up") label

Extremely versatile tool Currently state of the art (especially lentiviral vectors) Can also serve as model for experimental cell based gene therapies

Long standing tool in developmental biology

Signal amplification when enzymatic reaction is applied Can also be detected with antibody against (-gal

Fewer animals necessary for time series experiments Detected cells must be alive

Stable integration of the transcript Specific expression in certain cells if FP

under the control of a specific promotor Selection of altered clones less likely than in viral transduction of cells "Last resort" if no other method reliable enough or feasible

Relatively cheap

No additional labeling procedures necessary

Frequently problems with transgene inactivation in vivo

Cell properties may be changed by transformation/

viral integration Lab has to be set up to be able to handle viruses

LacZ antigen needs special fixation method to be detectable with an antibody See also viral transduction of cells with genes encoding fluorescent proteins In vivo tracking is not yet possible

Only clusters of cells can be traced For histological studies co-label (e.g. FP) necessary See also viral transduction of cell with viral transduction of cells with genes encoding fluorescent proteins

Only applicable for cell preparations from some animals, i.e. rat, mouse, pig

Difficult detection method that is not established in many labs (in situ hybridization) Difficult co-stains Immunological rejection

Antibody based detection has to be established first Most of the time, antigen retrieval is necessary Sometimes inconsistent staining results, appropriate controls are necessary t/5 t fD

fD B

mouse

Detecting DiI-labeled cells in cryosections:

1. Let the cryosections air-dry for 30-60 min at room temperature. During the procedure protect the slides from direct light.

2. Hydrate the slides in PBS for 5 min at room temperature.

3. Incubate in DAPI (4',6-diamidino-2-phenylindole, e.g. Sigma D9542) at a concentration of 1 pg/mL in dd H2O for 3-5 min at room temperature.

4. Wash twice in PBS for 5 min each.

5. Coverslip the slides with fluorescent mounting medium (e.g. DAKO). Histological analysis should be performed within the next 3 days. LacZ labeling

NSCs can be transfected or transduced with vectors coding for markers such as fluorescent proteins or LacZ, so that they can be distinguished from the host cells.

Expression of LacZ must be confirmed in the cells designated for transplant action, since transgenes can be downregulated during extended culture. During the last split before transplantation, an extra dish of cells should be set aside for X-gal processing. This dish should exhibit at least 75-90% blue cells. If the percentage is less than this, it is best to abort the transplant and thaw another earlier passage of the cell line.

LacZ detection by X-gal staining:

1. Euthanize transplanted animals by an overdose of pentobarbital (100 mg/kg) and prepare by transcardiac perfusion with 4% buffered paraformaldehyde (PFA).

2. Dissect brain.

3. Fix with 4% PFA overnight.

4. Incubate in 10% sucrose overnight.

5. Incubate in 30% sucrose overnight.

6. Blot the excess solution with Kimwipe.

7. Quick-freeze brains on dry ice.

8. Cut brains with razor blade and put slices onto microscope slides. Alternatively, brains can be cryosectioned at 20 pm.

9. Wash slices with 1X PBS at room temperature for 3 min.

10. Rinse with Solution A twice at room temperature for 10 min.

11. Permeabilize tissue in Solution C twice at room temperature for 10 min.

12. Finish preparation of Solution B: add 50 pL X-gal to 2.5 mL, add to tissue, keep covered in aluminum foil.

13. Incubate at 37°C overnight in box covered with aluminum foil (keep moist).

14. Visualize blue color (Figure 23.2) with bright field microscope.

Lateral Ventricle Injection
Figure 23.2 LacZ-expressing neural progenitors that have migrated throughout the brain after injection into a lateral ventricle.

Was this article helpful?

0 0
Addiction To Nutrition

Addiction To Nutrition

Get All The Support And Guidance You Need To Be A Success At Beating Addictions With Nutrition. This Book Is One Of The Most Valuable Resources In The World When It Comes To A Definitive Guide To Unchain Addiction The Smarter And Healthy Way.

Get My Free Ebook


Responses

  • Freya
    What is dil in labelling the embryo?
    8 years ago

Post a comment