Materials and reagents

Biological materials

Animals

Mice: Day-0 mice pups, C57BL/6 (Strain #000664, common name B6) obtained from The Jackson Laboratory

Viruses: RSA59, an isogenic recombinant strain of murine beta corona virus (MHVA59) [23]

Reagents

1. Hank’s balanced salt solution (HBSS) (10×) (Gibco, catalog number: 14185-052)

2. Phosphate buffered saline (PBS) pH 7.4 (10×) (Gibco, catalog number: 70011-044)

3. Sodium bicarbonate (NaHCO₃) (7.5%) (Gibco, catalog number: 25080-094)

   CAUTION: NaHCO₃ is skin and eye irritant. Wear suitable gloves, goggles, and protective clothing.

4. Dulbecco’s modified Eagle medium (DMEM) (powder) (Gibco, catalog number: 12100-046)

5. Trypsin 0.25% (1×) (Gibco, catalog number: 15050-057)

6. L-Glutamine 200 mM (100×) (Gibco, catalog number: 25030-081)

7. 0.5% Trypsin-EDTA (10×) (Gibco, catalog number: 15400-054)

   CAUTION: Components containing Trypsin may produce an allergic reaction. EDTA may cause irritation to the eyes and skin. Use respiratory protection as well as protection for skin and eyes.

8. Heat inactivated horse serum (origin: New Zealand) (Gibco, catalog number: 26050-070)

9. Penicillin-streptomycin (Pen-Strep) (Gibco, catalog number: 15140-122)

10. Distilled water (Gibco, catalog number: 15230-147)

11. Sterile deionized H₂O

12. F12 nutrient mixture (Ham) medium (Gibco, catalog number: 11765-054)

13. Heat inactivated fetal bovine serum (FBS) (Gibco, catalog number: 10082-147)

14. D-(+)-Glucose, Hybri-Max, ≥99.5% (Sigma-Aldrich, catalog number: G5146)

15. Trypsin from bovine pancreas (Sigma-Aldrich, catalog number: T9201-5G)

16. Laminin from Engelberth-holm swarm murine sarcoma basement membrane (Sigma-Aldrich, catalog number: L2020-1MG)

17. MEM non-essential amino acids solution (Sigma-Aldrich, catalog number: M7145)

18. HEPES buffer (1 M) (Gibco, catalog number: 15630-080)

    CAUTION: It may cause irritation to the skin, eyes, and upper respiratory tract. Wear protective gloves, goggles, and mask.

19. Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I6634-50MG)

20. Poly-D-Lysine hydrobromide (Sigma-Aldrich, catalog number: P7886-100MG)

21. Neurobasal medium (NB) (Gibco, catalog number: 21103-049)

22. Deoxyribonuclease I (DNase I) from bovine pancreas (Sigma-Aldrich, catalog number: D5025-15KU)

    CAUTION: May cause allergy or asthma symptoms or breathing difficulties if inhaled. Use protective clothing, gloves, and masks while using. Do not vortex, as DNase I is prone to physical denaturation.

23. B27 supplement 50× (Gibco, catalog number: 17504-044)

24. Magnesium chloride (MgCl₂·6H₂O) (SISCO Research Laboratories PVT LTD, catalog number: 1349130)

25. Calcium chloride (CaCl₂·2H₂O) (SRL, catalog number: 10035-04-8)

26. L-Thyroxine sodium salt pentahydrate (T4 thyroxine) (Sigma-Aldrich, catalog number: T0397-1MG)

27. Apo-Transferrin human (transferrin) (Sigma-Aldrich, catalog number: T-4382)

28. Putrescine dihydrochloride (Sigma-Aldrich, catalog number: P5780-5G)

29. Progesterone (Sigma-Aldrich, catalog number: P8783-5G)

30. Selenium dioxide (Sigma-Aldrich, catalog number: 325473)

    CAUTION: Toxic if swallowed or inhaled. May cause damage to organs through prolonged or repeated exposure. Very toxic to aquatic life with long-lasting effects. Avoid contact with skin, eyes, and clothing. Use a face shield, safety glasses, gloves, and respiratory protectant.

31. Biotin (Sigma-Aldrich, catalog number: B-4501)

32. 0.15 M Borate (Sigma-Aldrich, catalog number: B6768-1KG)

33. 99.9% Ethanol, absolute, analytical CSS reagent (Changshu song sheng fine chemicals, catalog number: GB678-90)

    CAUTION: Highly flammable liquid may cause drowsiness and nausea.

34. Sodium chloride (NaCl) (SRL, catalog number: 7647-14-5)

35. Potassium chloride (KCl) (MERCK, catalog number: 7447-40-7)

36. di-Sodium hydrogen phosphate (Na₂HPO₄) (MERCK, catalog number: 7558-79-4)

37. Potassium dihydrogen phosphate (KH₂PO₄) (MERCK, catalog number: 7778-77-0)

38. Trypan Blue (0.4%) (Gibco, catalog number: 15250061)

Growth factors

1. Nerve growth factor (NGF) (Invitrogen, catalog number: 13257-019)

2. Fibroblast growth factor (FGF) (R&D System, catalog number: 133-FB/CF)

3. Platelet-derived growth factor (PDGF) (R&D System, catalog number: 221-AA)

4. Neurotrophin-3 (NT3) (PeproTech, catalog number: 450-03-10UG)

Reagents for immunofluorescence

1. Triton X-100 (Sigma-Aldrich, catalog number: T8787-100ml)

   CAUTION: Harmful if swallowed. Causes skin irritation and serious eye damage. Use skin and eye protectants.

2. Bovine serum albumin, for molecular biology (BSA) (HIMEDIA, catalog number: MB083-25G)

   CAUTION: Causes skin and eye irritation; may cause an allergic response. Avoid contact with skin and eyes and wear respiratory protection, gloves, and goggles.

3. Antibodies

   Primary antibodies against various cell markers are diluted with 0.1% BSA (prepared from 1% BSA) as per the given ratio (Table 1).

   
   

   Table 1. Primary antibodies

   Antibody name	Dilution	Cellular markers for	Recommended storage condition
   Mouse anti-GFAP (Sigma, catalog number: G3893)	1:100	Astrocytes	Stored at -20 °C freezer
   Rabbit anti-Vimentin (CST, catalog number: D21H3)	1:200	Meningeal fibroblasts
   Rabbit anti-Iba1 (SIGMA Wako, catalog number: 019-19741)	1:600	Microglia
   Mouse anti-NG2 (MERCK, catalog number: MAB5384-I)	1:50	OPCs
   Rabbit anti-MAP 2 (SIGMA, catalog number: M3696)	1:200	Neurons
   Mouse anti-NFM (SIGMA, catalog number: N5389)	1:100	Neurons
   Mouse anti-Gal C (H8H9) (Homemade)	1:20	Mature oligodendrocytes
   Mouse anti-A2B5 (Homemade)	1:20	OPCs
   Rabbit anti-Cx43 (SIGMA, catalog number: C6219)	1:1,000	a gap junction protein

   Fluorescently tagged secondary antibodies raised against mouse or rabbit are diluted in 0.1% BSA (prepared from 1% BSA) as per the given ratio (Table 2).

   
   

   Table 2. Secondary antibodies

   Antibody name	Dilution	Recommended storage condition
   Donkey anti-Rabbit Alexa Fluor 568 (Invitrogen, catalog number: A10042)	1:1,000	Stored at 4 °C
   Goat anti-Mouse Alexa Fluor 488 (Invitrogen, catalog number: A11001)	1:1,000
   Goat anti-Mouse Alexa Fluor 546 (Invitrogen, catalog number: A11003)	1:1,000

Solutions

1. DMEM solution (see Recipes)

2. Astrocyte-specific medium (see Recipes)

3. Hanks for prep solution (see Recipes)

4. Serum for prep solution (see Recipes)

5. 1× PBS (see Recipes)

6. HBSS solution (see Recipes)

7. NB/B27 medium (see Recipes)

8. Oligodendrocytes-specific media (see Recipes)

9. Neuron-specific media (see Recipes)

10. DM supplement (see Recipes)

11. T4 thyroxine (see Recipes)

12. 30% glucose (1.66 M) (see Recipes)

13. Insulin (see Recipes)

14. Biotin (see Recipes)

15. Oligodendrocyte differentiation media (see Recipes)

16. DMEM Minus (-) (see Recipes)

17. DMEM Plus (+) (see Recipes)

18. HBSS (+) or 1% glucose HBSS solution (see Recipes)

19. DNase I solution (see Recipes)

20. Trypsin (see Recipes)

21. Poly-D-Lysine (PDL) coating (see Recipes)

22. Wash buffer (see Recipes)

23. Blocking serum (1% BSA) (see Recipes)

Recipes

1. DMEM solution

   Prepare by dissolving DMEM powder in 1 L of double-autoclaved Milli Q water; then, add NaHCO₃. Follow the table below for preparation. Mix properly (so that no clumps remain) and then adjust the pH to 7.2–7.4. Sterilize the solution by passing through a 0.22 µm filter paper. The solution can be stored at 4 °C for up to one month.

   Components	Quantity	Final concentration	Recommended storage condition
   DMEM powder	1 pouch	13.4 g/L	4 °C
   NaHCO3	3.7 g/L	44.04 mM	Room temperature (20–25 °C)
   Double-autoclaved Milli Q H2O	1 L	NA	Room temperature
   Final volume	1 L		4 °C




2. Astrocyte-specific medium

   Sterilize the solution by passing through a 0.22 µm filter paper. Always prepare fresh; the solution can be stored for approximately one month at 4 °C.

   Components	Quantity	Final concentration	Recommended storage condition
   FBS	10 mL	10% (v/v)	-20 °C
   Pen-Strep	1 mL	1% (v/v)	-20 °C
   L-Glutamine	0.1 mL	0.2 mM	-20 °C
   DMEM solution	89.9 mL	NA	4 °C
   Final volume	100 mL		4 °C




3. Hanks for prep solution

   Sterilize the solution by passing through a 0.22 µm filter paper before use. The solution can be stored at 4 °C for up to 1–2 weeks.

   Components	Quantity	Final concentration	Recommended storage condition
   HBSS (10×)	10 mL	10% (v/v)	4 °C
   1 M HEPES buffer	2.5 mL	25 mM	4 °C
   7.5% NaHCO3	1 mL	1% (v/v)	4 °C
   Pen-Strep	1 mL	1% (v/v)	-20 °C
   Double-autoclaved Milli Q H2O	85.5 mL	NA	Room temperature
   Final volume	100 mL		4 °C




4. Serum for prep solution

   Sterilize the solution by passing through a 0.22 µm filter paper before use. The solution can be stored at 4 °C for up to one week.

   Components	Quantity	Final concentration	Recommended storage condition
   Heat-inactivated FBS	10 mL	10% (v/v)	-20 °C
   Non-essential amino acid solution	1 mL	1% (v/v)	4 °C
   L-Glutamine	1 mL	2 mM	-20 °C
   Pen-Strep	1 mL	1% (v/v)	-20 °C
   DMEM	87 mL	NA	4 °C
   Final volume	100 mL		4 °C




5. 1× PBS

   Dissolve the following reagents as per the given quantities in 800 mL of distilled water and adjust the pH to 7.2–7.4; then, make up the volume up to 1 L and filter sterilize the solution by passing through a 0.22 µm filter paper.

   Components	Quantity	Final concentration	Recommended storage condition
   NaCl	8 g	137 mM	Room temperature
   KCl	0.2 g	2.7 mM	Room temperature
   Na2HPO4	0.24 g	10 mM	Room temperature
   KH2PO4	1.44 g	1.8 mM	Room temperature
   Distilled water	1 L	NA	Room temperature
   Final volume	1 L		4 °C




6. HBSS solution

   100× Ca²⁺ (90 mM)

   Dissolve 1.324 g of CaCl₂·2H₂O in 100 mL of distilled water.

   
   

   100× Mg²⁺ (104.8 mM)

   Dissolve 2.13 g of MgCl₂·6H₂O in 100 mL of distilled water.

   
   

   HBSS with Ca²⁺ and Mg²⁺

   Sterilize the solution by passing through a 0.22 µm filter paper. This solution can be stored for up to 1–2 months at 4 °C.

   Components	Quantity	Final concentration	Recommended storage condition
   HBSS (10×)	10 mL	10% (v/v)	4 °C
   Ca2+ (100×)	1 mL	0.9 mM	4 °C
   Mg2+ (100×)	1 mL	1.05 mM	4 °C
   Double-autoclaved Milli Q H2O	88 mL	NA	Room temperature
   Final volume	100 mL	1% (v/v)	4 °C




7. NB/B27 medium

   Sterilize the solution by passing through a 0.22 µm filter paper. Always prepare fresh and do not store the solution for more than two weeks.

   Components	Quantity	Final concentration	Recommended storage condition
   B27 Supplement 50×	2 mL	2% (v/v)	4 °C
   Pen-Strep	2.5 mL	2.5% (v/v)	-20 °C
   L-glutamine	2.5 mL	5 mM	-20 °C
   NB	93 mL	NA	4 °C
   Final volume	100 mL		4 °C




8. Oligodendrocytes-specific media

   Always prepare fresh and use it according to your experimental planning.

   Critical step: Do not filter the growth factors. Always add the growth factors instantly in the NB/B27 incomplete media just before use for your experiments and put growth factors back in the -20 °C freezer without any delay.

   Components	Quantity	Final concentration	Recommended storage condition
   PDGF (1 µg/mL)	100 µL	2 ng/mL	-20 °C
   FGF (10 µg/mL)	50 µL	10 ng/mL	-20 °C
   NT3 (1 µg/mL)	50 µL	1 ng/mL	-20 °C
   NB/B27 medium	50 µL	NA	4 °C
   Final volume	50 µL		4 °C




9. Neuron-specific media

   Always prepare the solution fresh just before the experiment.

   Critical step: Do not filter the growth factors. Caution should be followed as aforesaid for oligodendrocytes-specific medium.

   Components	Quantity	Final concentration	Recommended storage condition
   NGF (100 µg/mL)	7 µL	50 ng/mL	-20 °C
   NB/B27 medium	15 mL	NA	4 °C
   Final volume	mL		4 °C




10. DM supplement

    Filter sterilize the mixture after combining all components using a 0.2 µm filter. Store 1 mL aliquots at -20 °C for approximately two months.

    Components	Quantity	Final concentration	Recommended storage condition
    Transferrin	5 mL	250 mg/5 mL DMEM	-20 °C
    Putrescine	5 mL	26.85 mg/5 mL DMEM (33.3 mM)	Room temperature
    Progesterone	10 mL	3 mg/5 mL [of 95% EtOH (1.908 mM)] (dilute 25 µL of this in 10 mL DMEM)	Room temperature
    Selenium dioxide	5 mL	3.3 mg/10 mL DMEM (2.974 mM) (dilute 40 µL of this in 5 mL DMEM)	Room temperature
    Final volume	25 mL		-20 °C




11. T4 thyroxine

    Aliquot and store at 4 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    T4 thyroxine	1 mg	22.5 µM	-20 °C
    DMEM	50 mL	NA	4 °C
    Final volume	50 mL		4 °C




12. 30% glucose (1.66 M)

    Dissolve 30 g of glucose in 100 mL of sterile ddH₂O. Sterilize the solution by passing through a 0.22 µm filter paper and store at 4 °C.




13. Insulin

    Mix the provided quantity of insulin in DMEM and then add 1 N HCl dropwise until the cloudy solution clears. Filter sterilize the solution and store 1 mL aliquots at 4 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    Insulin	25 mg	4.36 µM	-20 °C
    DMEM	10 mL	NA	4 °C
    1 N HCl	NA	NA	Room temperature
    Final volume	10 mL		4 °C




14. Biotin

    Dissolve biotin in doubled-distilled water (ddH₂O) (5 mg in 100 mL of ddH₂O) to make a stock of 204.65 µM. Then, prepare a working stock of 40.93 µM as per the instructions below and sterilize the solution by passing through a 0.2 µm filter paper; store at 4 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    Biotin	20 mL	40.93 µM	4 °C
    DD H2O	80 mL	NA	Room temperature
    Final volume	100 mL		4 °C




15. Oligodendrocyte differentiation media

    Mix all the given components and store the solution at 4 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    T4 thyroxine	4 mL	0.9 µM	-20 °C
    30% glucose	2 mL	33.4 mM	4 °C
    L-Glutamine	2 mL	4 mM	-20 °C
    Pen-Strep	2 mL	2% v/v	-20 °C
    Insulin	1 mL	4.36 µM	4 °C
    DM supplement	1 mL	1% v/v	-20 °C
    Biotin	200 µL	81.86 µM	4 °C
    DMEM	94 mL	NA	4 °C
    F12 medium	94 mL	NA	4 °C
    Final volume	200 mL		4 °C




16. DMEM Minus (-)

    Sterilize the solution by passing through a 0.22 µm filter paper.

    Critical: Always prepare fresh and store at 4 °C for no more than 1–2 weeks.

    Components	Quantity	Final concentration	Recommended storage condition
    1 M HEPES buffer	1 mL	10 mM	4 °C
    Pen-Strep (100×)	1 mL	1% (v/v)	-20 °C
    Non-essential amino acid solution	1 mL	1% (v/v)	4 °C
    DMEM	97 mL	NA	4 °C
    Final volume	100 mL		4 °C




17. DMEM Plus (+)

    Sterilize the solution by passing through a 0.22 µm filter paper.

    Critical: Prepare fresh and store at 4 °C refrigerator for not more than 1–2 weeks. FBS and HS should be filtered first to prevent the clogging of filter paper.

    Components	Quantity	Final concentration	Recommended storage condition
    1 M HEPES buffer	1 mL	10 mM	4 °C
    Pen-Strep (100×)	1 mL	1% (v/v)	-20 °C
    Non-essential amino acid solution	1 mL	1% (v/v)	4 °C
    Heat inactivated FBS	5 mL	5% (v/v)	-20 °C
    Heat inactivated horse serum	5 mL	5% (v/v)	-20 °C
    DMEM	87 mL	NA	4 °C
    Final volume	100 mL		4 °C




18. HBSS (+) or 1% glucose HBSS solution

    Sterilize the solution by passing through a 0.22 µm filter paper. The solution can be stored for up to one month at 4 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    HBSS (1×)	100 mL	NA	4 °C
    Glucose	1 g (w/v)	55.5 mM	Room temperature
    Final volume	100 mL		4 °C




19. DNase I solution

    DNase I enzyme is commercially available as a powder. Sterilize the solution by passing through a 0.2 µm filter paper before use. Aliquot the solution to 2 mL tubes and store at -20 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    DNase I	3 mg	0.1 mg/mL	-20 °C
    1× PBS	30 mL	NA	4 °C
    Final volume	30 mL		-20 °C




20. Trypsin

    Sterilize the solution by passing through a 0.2 µm filter before use. Aliquot the solution in 2 mL tubes and store at -20 °C.

    Components	Quantity	Final concentration	Recommended storage condition
    Trypsin from bovine pancreas	300 mg	10 mg/mL	-20 °C
    1× PBS	30 mL	NA	4 °C
    Final volume	30 mL		-20 °C




21. Poly-D-Lysine (PDL) coating

    Dissolve 5 mg of PDL in 100 mL of distilled water, then add 400 mL of distilled water to make up the volume.

    Sterilize the solution by passing through a 0.2 µm filter paper. Store at 4 °C.

    To coat:

    Add 1 mL of 0.15 M borate (in 0.15 M NaOH, pH 8.4) per 100 mL of PDL solution and filter coat flasks with the PDL-borate for at least 2 h at room temperature.

    Suction the liquid off the plastic and dry at 37 °C overnight.

Reagent setup for immunofluorescence

1. Wash buffer

   Critical: Always prepare fresh just before using and store at 4 °C.

   Components	Quantity	Final concentration	Recommended storage condition
   1× PBS	49 mL	NA	4 °C
   Ca2+ (100×)	0.5 mL	0.9 mM	4 °C
   Mg2+ (100×)	0.5 mL	1.04 mM	4 °C
   Final volume	50 mL		4 °C




2. Blocking serum (1% BSA)

   Mix properly using the vortex and keep the solution for some time to settle the frothing.

   Critical: Do not vortex for a long period, otherwise it will cause too much frothing in the solution.

   Instead of BSA, goat serum can also be used for the preparation of blocking serum and antibody diluents.

   Components	Quantity	Final concentration	Recommended storage condition
   BSA	1 g	1% (w/v)	4 °C
   Wash buffer	100 mL	NA	4 °C
   Final volume	100 mL		4 °C

Laboratory supplies

1. Cell strainer (70 and 100 μm) (Falcon, catalog numbers: 352350 and 352360)

2. 15 mL centrifuge tubes (Tarsons, catalog number: 546021)

3. 50 mL centrifuge tubes (Tarsons, catalog number: 546041)

4. Serological pipette (5 mL and 10 mL) (Thermo scientific Nunc, catalog numbers: 170355N and 7128)

5. 6-well plate (Nunc Thermo, catalog number: 140675)

6. 35 mm disc (Nunc Thermo Scientific, catalog number: 150460)

7. 60 mm disc (Nunc Thermo Scientific, catalog number: 150288)

8. 100 mm disc (Nunc Thermo Scientific, catalog number: 150350)

9. Bacterial Petri dish (Tarson, catalog number: 460095)

10. Pipette controller (Gilson, catalog number: 101300)

11. Syringes (Dispo Van, Hindustan Syringes and Medical devices, catalog number: 840054SM1)

12. Pipette tips [Tarson, catalog numbers: 521020 (200–1,000 µL), 521010 (2–200 µL), 521000 (0.2–10 µL)]

13. 0.22 µm filter (Merck, Life Sciences, catalog number: B20345)

14. 4 chambers (mounted on permanox) slides (Nunc, catalog number: 120075LE 1007)

Equipment

1. Biosafety hood (Thermo Scientific, Heraguard, catalog number: 41237696)

2. Centrifuge (Eppendorf, model: 5702R, catalog number: 5703YH207703)

3. CO₂ incubator (Thermo Scientific, model: Heracell 150i, catalog number: G-3168)

4. Dissection microscope (Nikon SMZ 745 Model C-LEDS, catalog number: 226763)

5. Microscope (Nikon, model: Eclipse Ts2, catalog number: 137714)

6. Analytical balance (Sartorius PRACTUM213-10IN, catalog number: S/N 003670729)

7. Shaker incubator (ZHICHENG, catalog number: G-1377 ZHWY-103B)

8. Water bath with shaker (Jio Tech, catalog number: G-3179)

9. Forceps (Fine Science Tools, catalog number: 00649-11)

10. Fine forceps (Fine Science Tools, catalog numbers: 11251-33, 11252-40, 11252-30)

Software and datasets

For image processing

1. ImageJ (NIH, USA)

2. Zen 2010 software (Carl Zeiss, Germany)

Procedure