Dual Fluorescence Imaging to Identify Differentiated Oligodendrocytes

Abstract

Source: Schott, J. T., et al. Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning. J. Vis. Exp. (2016).

This video demonstrates the use of dual fluorescence imaging to evaluate the differentiation of oligodendrocytes from precursor cells (OPCs) isolated from a rat pup brain. The process includes fixing the cells, labeling specific markers with primary and fluorophore-conjugated secondary antibodies, and visualizing them using fluorescence imaging. Differentiated oligodendrocytes expressing both myelin basic protein (MBP) and oligodendrocyte transcription factor 2 (Olig2) are identified, distinguishing them from undifferentiated precursor cells that express only Olig2.

Protocol

1. Preparation of Assay Plates, Stock Solutions, and OPC Base Media

Note: The rat OPC base (Sato) media described here has been derived from previously published studies. Alternative media formulations may also be compatible with this procedure.

1. Resuspend poly-L-lysine (PLL) to a concentration of 10 µg/ml in sterile deionized water. Pipette 400 μl of the diluted PLL into each well of a black-walled, clear bottom 24 well tissue culture grade dish.
2. Coat tissue culture dishes for 2 hr in a 37 °C tissue culture incubator or overnight in a 4 °C refrigerator.
3. Aspirate PLL from coated dishes at least 20 min prior to plating. Allow the remaining PLL to dry from the well by placing 24 well plates with the lid partially ajar in a tissue culture hood. Verify that the wells are completely dry before plating isolated OPCs. Cells will not adhere to plastic that has liquid PLL present.
4. Prepare N-Acetyl-L-cysteine (NAC) stock solution (1,000x): Dissolve 100 mg N-Acetyl-L-cysteine (NAC) in 20 ml of Dulbecco's modified Eagle's medium (DMEM). Aliquot and store at -20 °C.
5. Prepare Hydrocortisone stock solution (1,000x): Add 1 ml of ethanol to 1 mg Hydrocortisone and swirl to dissolve. Add 19 ml of DMEM, mix the solution, aliquot and store at -20 °C. The addition of hydrocortisone to the base media has been shown to enhance the survival of glial cells.
6. Prepare d-Biotin stock solution (5,000x): Dissolve 2.5 mg of d-biotin in 50 ml of phosphate-buffered saline (PBS). Add 1-2 x 5 µl drops of 0.1 N NaOH to aid in dissolution. Aliquot and store at -20 °C.
7. Prepare Insulin stock solution (100x): Dissolve 25 mg Insulin in 50 ml of tissue culture grade water. Add 250 µl of 1 N HCl and mix until solution is clear. Sterilize with a 0.22 µm filter and store at 4 °C for up to 6 weeks.
8. Prepare Sato stock solution (100x):
   1. Prepare progesterone stock solution (25 µg/µl): Dissolve 2.5 mg progesterone in 100 µl of ethanol.
   2. Prepare sodium selenite stock solution (400 ng/µl): Dissolve 4 mg sodium selenite in 100 µl of 0.1 N NaOH. Add 10 ml of DMEM and mix.
   3. To 100 ml of DMEM, add 1 g of human apo-Transferrin, 1 g of bovine serum albumin, and 160 mg of putrescine and mix to fully dissolve. Add 25 µl of progesterone stock solution, 1,000 µl of sodium selenite stock solution, and mix. Aliquot and store at -20 °C.
9. Prepare OPC Base Media: per 100 ml of DMEM (with 4.5 g/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate), add 100 µl NAC, 100 µl hydrocortisone, 20 µl d-Biotin, 1 ml insulin, 1 ml Sato stock, 100 µl trace elements B, 2 mL B-27 supplement (50x), and 1 ml of penicillin/streptomycin (100x). Sterilize with a 0.22 µm filter and store at 4 °C.
10. Prepare platelet-derived growth factor AA (PDGF-AA) stock solution (1,000x): Dissolve 250 μg in 12.5 ml of PBS with 0.1% bovine serum albumin (BSA) to make a 20 μg/ml solution. Aliquot and store at -80 °C.
11. Prepare OPC proliferation media: OPC base media supplemented with 20 ng/ml PDGF-AA.
12. Prepare OPC differentiation media: OPC base media supplemented with 45 nM triiodothyronine.
13. Prepare Column Buffer: To 500 ml of PBS, add 2.5 g of BSA and 2 ml of 0.5 M ethylenediaminetetraacetic acid (EDTA) and adjust the pH to 7.2. Sterilize with a 0.22 µm filter and store at 4 °C.

2. Induction of OPC Differentiation and Fixation with 4% Paraformaldehyde

Note: When testing the effect of small molecules on oligodendrogenesis, we routinely dissolve drugs in dimethyl sulfoxide (DMSO) to prepare 1,000x stocks that can then be stored at -80 °C and diluted directly into SATO media to obtain a final concentration of 0.1% DMSO. We have observed no changes in either OPC proliferation or differentiation using this concentration of DMSO (data not shown).

1. Pre-warm OPC base media to 37 °C and thaw drug treatment stocks to room temperature. When performing treatments in duplicate, prepare approximately 1.25 ml of media per treatment group in a 1.5 ml tube. For triplicate samples, use 2 ml capacity centrifuge tubes to account for slack.
2. Prepare the treatment master mixes for replicate wells by diluting treatment stocks directly into OPC base media. Briefly vortex or gently mix each mastermix to ensure homogeneous distribution of the treatment.
3. Prepare 45 nM triiodothyronine (T₃) as the positive control and the appropriate vehicle as the negative control. Dilute 10 mM T₃ stock 1:1,000 in 1 ml of OPC base media and then further dilute in the treatment master mix to reduce the final concentration of DMSO if necessary.
4. Aspirate the media from the culture wells one treatment group at a time so as to avoid drying the cells. Use a 200 µl pipet tip on the end of the glass Pasteur pipet to avoid scraping off black material from the sides of the well and into the culture.
5. Slowly add 500 µl of treatment mastermix along the side of the well using a 1 ml pipet.
6. Incubate the cultures for the desired timeframe, performing a full media exchange with fresh treatment media every 2-3 days. We routinely observe a 30-40% MBP⁺ culture after 4 days in differentiation media.
7. At the end of the desired treatment period, prepare fresh 4% paraformaldehyde (PFA) or thaw a frozen stock to room temperature. CAUTION: PFA is extremely toxic and must be prepared in a fume hood.
8. Aspirate the media and slowly add 400 µl of PFA to the side of the well to fix the cells. Incubate the plate for 20 min at room temperature.
9. Aspirate the PFA and slowly add 500 µl of sterile Dulbecco's phosphate-buffered saline (D-PBS, with Ca²⁺ and Mg²⁺) to the side of the well to wash the cells. Repeat the wash a total of two more times. Do not aspirate the final wash.
10. Wrap the plate in parafilm and store at 4 °C for later processing or proceed directly to the next step. Plates can be stored for several weeks.

3. Immunocytochemistry and Quantification of Myelin Basic Protein

Note: When performing liquid handling procedures on the 24 well plates, it is recommended to work fast to avoid drying the cells. Here, the use of a multi-channel vacuum aspirator and multi-channel pipet are recommended.

1. Bring the plate to room temperature, aspirate the wells, and add 250 μl of D-PBS (with Ca²⁺ and Mg²⁺) containing 0.1% Triton X-100 and 5% normal goat serum (NGS). Incubate the plate at room temperature for 1 hr with gentle orbital shaking.
2. Prepare the primary incubation solution by diluting mouse monoclonal anti-MBP antibody 1:1,000 and rabbit polyclonal anti-Actin antibody 1:200 in D-PBS (with Ca²⁺ and Mg²⁺) containing 5% NGS. Alternatively, rabbit polyclonal anti-Olig2 at 1:1,000 can be used for oligodendrocyte specific normalization in mixed glial cultures. Prepare enough primary solution to accommodate 250 µl per well.
3. Incubate primary antibodies at 4 °C overnight for 16-18 hr with gentle orbital shaking.
4. Aspirate the primary incubation solution and wash the cells 3 x 10 min with 500 µl of D-PBS (with Ca²⁺ and Mg²⁺) at room temperature with gentle orbital shaking.
5. While washing the plate, prepare the secondary incubation solution by diluting anti-mouse 680 and anti-rabbit 800 antibodies 1:500 in D-PBS (with Ca²⁺ and Mg²⁺) containing 5% NGS. Minimize ambient light exposure when preparing this solution.
6. Aspirate the final wash and add 250 µl of secondary incubation solution. Protect the plate from light and incubate it at room temperature for 1 hr with gentle orbital shaking.
7. Aspirate the secondary incubation solution and wash the wells 3 x 10 min with 500 µl of D-PBS (with Ca²⁺ and Mg²⁺) at room temperature with gentle orbital shaking.
8. Scan the plate using an imaging system capable of detecting 700 nm and 800 nm fluorescence emissions. As a starting point, set the focal offset to 3 and sensitivities to 1.5 for MBP, 5.0 for Actin, and 3.5 for Olig2. Adjust the sensitivity values as needed to avoid signal overexposure.
9. Quantify the extent of oligodendrogenesis using semi-automated software-based methods.
   1. Using the software, set the plate analysis mode to "24 Well" and align circles around the outer perimeter of the scanned wells. Set the normalization channel to "800" and export the total and relative fluorescence intensities for the 700 nm (MBP) and 800 nm (Olig2/Actin) channels.
   2. Divide the intensity at 700 nm by the intensity at 800 nm to give the normalized MBP expression in relative fluorescence units. Calculate the average of replicate measurements and scale the expression to the Day 0 +PDGF controls as needed for the analysis.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Culture Materials
PDGF-AA	PeproTech Inc	100-13A	20 ng/mL
Dimethyl sulfoxide	Sigma-Aldrich	D2650	Hybri-Max 100 mL
Triiodothyronine	Sigma-Aldrich	T6397	45 nM
Clemastine fumarate salt	Sigma-Aldrich	SML0445-100MG	1 μM
Quetiapine hemifumarate salt	Sigma-Aldrich	Q3638-10MG	1 μM
N-acetyl cysteine	Sigma-Aldrich	A9165	5 μg/mL
Progesterone	Sigma-Aldrich	P8783	60 ng/mL
Poly-L-lysine	Sigma-Aldrich	P1524	10 μg/mL
Putrecine	Sigma-Aldrich	P7505	16 μg/mL
Sodium Selenite	Sigma-Aldrich	S5261	40 ng/mL
Hydrocortisone	Sigma-Aldrich	H0135	50 ng/mL
d-Biotin	Sigma-Aldrich	B4639	10 ng/mL
Insulin	Sigma-Aldrich	I6634	5 μg/mL
Apo-Transferrin	Sigma-Aldrich	T1147	100 μg/mL
Bovine Serum Albumin	Sigma-Aldrich	A4161	100 μg/mL
Trace Elements B	Corning	25-022-Cl	1 x
B-27 Supplement	Life Technologies	17504044	1 x
Penicillin/Streptomycin	Life Technologies	15140122	1 x
DMEM	Life Technologies	11995-065	1 x
24-well Black Visiplate with lid	Perkin Elmer	1450-605	Tissue culture grade
Immunocytochemistry and Quantification
PFA	Sigma-Aldrich	100-13A	4%
Triton X-100	Sigma-Aldrich	78787	0.10%
Normal Goat Serum	Jackson Immuno Research	005-000-121	5%
mouse monoclonal anti-MBP	Biolegend	SMI-99P	1:1000 Dilution
rabbit polyclonal anti-Actin	Santa Cruz Biotecnology	SC-7210	1:200 Dilution
rabbit polyclonal anti-Olig2	Millipore	AB9610	1:1000 Dilution
goat anti-mouse 680RD	Licor	926-68070	1:500 Dilution
goat anti-rabbit 800CW	Licor	926-32211	1:500 Dilution
Alexa Fluor 594 goat anti-mouse	Life Technologies	A11032	1:000 dilution
Alexa Fluor 488 goat anti-rabbit	Life Technologies	A11008	1:000 dilution
Prolong Gold antifade with DAPI	Life Technologies	P36931
DPBS with Ca2+ and Mg2+	Corning	21-030-CV
Licor Odyssey Clx Infared Imaging System	Licor