TITLE: Neurosphere Protocol - A rapid and detailed method for the isolation of spheres in-vitro
AUTHORS: Christopher Blackwood

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The neurosphere assay is a powerful </span><span style = "font-style:italic;">in-vitro </span><span>tool that has been used to study neural stem cell biology. Here we demonstrate a rapid and detailed isolation approach that produces large number of neurospheres from the dorsal lateral ventricle of late embryonic mice without the need for specialized equipment. This approach can be used to generate neurospheres for genetic studies, pharmacological manipulation, immunocytochemistry, self-renewal and differentiation assays.</span></div></div>

[STEPS]
?. [Before you begin dissections]
1.	Set-up prior to embryo dissection
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1.1)	Establish breeding pairs of mice to obtain embryonic day 17 (E17) embryos.  Day 0 is defined as the day a vaginal plug is detected.
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1.2)	Prepare sterile surgical tools (scissors for decapitation, #5 forceps, razor blades).
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1.3)	Add 20 mls of Hank’s buffer to each of two 10 cm petri plates and place on ice.  Add 5 mls Hank’s buffer to a 15 ml tube and also place on ice. Reserve another 50 mls of room temperature Hank’s buffer.
?. [Before you begin dissections]
1.4)	Prewarm 10 mls of Hank’s-low BSA at 37 °C
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1.5)	Prewarm 5 mls of Hank’s-high BSA at 37 °C.
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1.6)	Prewarm 10 mls of DMEM/F12 with serum at 37 °C.
?. [Before you begin dissections]
1.7)	Prewarm 5-15 mls of neurosphere media at 37 °C. Amount is based on number of desired wells.
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1.8)	Prewarm 2 mls of 0.25% trypsin/EDTA at 37 °C.
?. [Tissue Dissection]
2.	Tissue dissection
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2.1)	Spray the abdomen with 70% ethanol, and make an incision to expose the uterus. Remove the uterus and transfer it to an empty petri plate.
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2.2)	Remove embryos from the uterus, spray desired number with 70% ethanol, and decapitate one or more embryos. Rinse each decapitated head in one petri plate containing Hank’s buffer, and then transfer to the second petri plate containing Hank’s buffer on ice.
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2.3)	Use forceps to remove the skin and skull. Remove the brain and place in an empty petri dish.
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2.4)	Separate the two hemispheres with a razor blade, and place one half of a brain on its lateral surface.
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2.5)	Using a stereomicroscope, identify the location of the lateral ventricle on the medial surface (the dorsal region of the lateral ventricle contains the dLGE).  The ventricle is visible as a T-shaped structure that is slightly darker than the rest of the brain. Using a razor blade or a scalpel, sequentially trim away the brain surrounding the ventricle on all four sides.
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2.6)	Transfer the dissected tissue into the 15 ml tube on ice.
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2.7)	If neurospheres are to be isolated from additional embryos (e.g. because of low yield), keep tube on ice until all dissections are complete.
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3.	Primary neurosphere culture
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3.1)	Spin sample at 300 RCF in a clinical centrifuge for 3 min. to pellet tissue.
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3.2)	Aspirate off the supernatant and add 2 mls of pre-warmed trypsin/EDTA. Incubate at 37 °C for 15 min. with intermittent swirling.
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3.3)	Spin tube at 300 RCF for 2 min.
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3.4)	Add 10 mls of room temperature Hank’s to trypsin/tissue mixture and incubate at 37 °C for 5 min. with intermittent swirling. Spin culture at 300 RCF for 3 min. and remove the supernatant.
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3.5)	Repeat wash step 3.4 two additional times.
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3.6)	Aspirate the supernatant and add 4 mls of Hank’s-low BSA.
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3.7)	Triturate the tissue gently and slowly approximately 10 times with an 18-gauge needle until tissue chunks appear relatively uniform in size. Avoid creating bubbles or foam.
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3.8)	Triturate the crude cell suspension gently and slowly approximately 7-10 times with a 21-gauge needle until tissue chunks appear relatively uniform in size.
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3.9)	Triturate the suspension approximately 4-5 times with a 23-gauge needle until suspension appears uniform.
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3.10) Add 3 mls of Hank’s-high BSA solution to a 15 ml tube. Slowly add the cell suspension to the bottom of the tube underneath the Hank’s-high BSA solution using a 23-gauge needle.
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3.11) Centrifuge at 300 RCF for 5 min.
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3.12) Aspirate supernatant and resuspend cells with 3 mls of prewarmed Hank’s-low BSA.
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3.13) Centrifuge at 300 RCF for 5 min.
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3.14) Aspirate supernatant, and resuspend cells in 5 mls of prewarmed DMEM/F12 with serum.
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3.15) Incubate tubes for 2-4 hours at 37 °C to reduce bacterial contamination.
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3.16) Centrifuge at 300 RCF for 5 min.
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3.17) Resuspend cells in 1 ml of prewarmed neurosphere media.
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3.18) Count cells with a hemocytometer. Plate 10,000 cells in a volume of 250 ul in each well of a 48-well plate. Plate at least 8 wells to ensure adequate numbers of neurospheres.
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3.19) Incubate at 37 °C in a humidified incubator with 5% CO2.
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3.20) Neurospheres should form within 3-4 days. At day 3, add an additional 100 ul of neurosphere media to each well.
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4.	Secondary neurosphere culture
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After 5-7 days, primary neurospheres should be split to maintain the health of the culture.
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4.1)	Prewarm 1 ml of Trypsin/EDTA at 37 °C.
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4.2)	Prewarm 10 mls of DMEM/F12 (without serum or antibiotics) at 37 °C.
?. [Secondary Neurosphere culture]
4.3)	Prewarm 2-15 mls of neurosphere media at 37 °C. Amount is based on number of desired wells.
?. [Secondary Neurosphere culture]
4.4)	Prewarm 1 ml of trypsin inhibitor at 37 °C.
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4.5)	Collect all neurospheres from at least 8 wells and transfer to a 15 ml tube.
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4.6)	Spin down neurospheres at 300 RCF for 5 min.
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4.7)	Remove supernatant and add prewarmed trypsin. Incubate at 37 °C for approximately 5-10 min., depending on the size of neurospheres.
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4.8)	Add 1 ml of trypsin inhibitor solution and swirl cells for 15 secs to inactivate trypsin.
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4.9)	Spin down culture at 300 RCF for 5 min.
?. [Secondary Neurosphere culture]
4.10) Resuspend cells in 4 mls of DMEM/F12.
?. [Secondary Neurosphere culture]
4.11) Triturate the crude cell suspension gently and slowly approximately 7-10 times with a 21-gauge needle until tissue chunks appear relatively uniform in size.
?. [Secondary Neurosphere culture]
4.12) Triturate the suspension approximately 4-5 times with a 23-gauge needle until suspension appears uniform.
?. [Secondary Neurosphere culture]
4.13) Spin down dissociated cells at 300 RCF for 5 min.
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4.14) Resuspend cells in 1 ml of DMEM/F12.
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4.15) Count cells using a hemocytometer, and plate 10,000 cells in a volume of 250 μl neurosphere media in each well of a 48-well plate. Plate at least 8 wells to ensure adequate numbers of neurospheres.
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4.16) Incubate at 37 °C in a humidified incubator with 5% CO2.