ab315364 – Human Spinal Motor Neurons (Male, WC-30) Imaging and 4.

4. As soon as the last of the ice melts, which will take ~75-90 seconds, remove the vial
from the water bath. Disinfect the vial by spraying it with 70% ethanol and transfer it to
Plate Reader Assays the cell culture hood.
For research use only - not intended for diagnostic use. 5. Slowly add 500 μL of seeding medium to the vial at a rate of ~1 drop/s using a 1 mL
pipette tip. This process should take about 30 seconds.
For overview, typical data and additional information please visit: 6. Gently transfer all contents (~1mL total) from the vial to a new sterile 50 mL conical
http://www.abcam.com/ab315364 tube.
7. To collect any residual cells, gently add another 1 mL of seeding medium to the vial
Storage and Stability and then gently transfer to the conical tube.
Immediately transfer the vial of neurons to liquid nitrogen upon receipt. Transfer the vials of 8. Slowly add an additional 3 mL of seeding medium to the 50 mL conical tube using a
supplements to a -20°C freezer. The supplements can be stored at -20°C for up to 6 months. 10 mL serological pipette. Gently swirl the conical tube while adding the medium. This
Alternatively, the supplements can be stored at -80°C for up to 18 months. process should take about 1 minute.
9. To count the cells, gently swirl the conical tube again and remove 10 μL from the cell
Materials Supplied suspension. Count the number of viable cells per mL with a hemocytometer using the
Item Quantity Storage Condition trypan blue exclusion method to identify dead/viable cells.
10. The recommended seeding density is 20,000 – 25,000 viable cells/well for a 96-well
One vial of 5 million or 1 million 500 µL -196ºC
plate (~65,000 – 78,000 viable cells/cm2). Use the following equation to determine the
cryopreserved human motor
volume of cell suspension needed for each 96-well plate:
neurons
BrainFast Motor Supplement 1 vial -20ºC or -80ºC
Volume of cell suspension needed (mL) = (2.4 – 3.0x106 cells)/(viable cells per mL).
(1000x)
BrainFast D4 Supplement (1000x) 1 vial -20ºC or -80ºC
11. In a separate 50 mL conical tube, add the calculated volume of cell suspension
BrainFast SK Supplement (1000x) 1 vial -20ºC or -80ºC needed, and then add enough medium to obtain a final volume of 12 mL. For
example, if the volume of cell suspension needed is 2 mL, combine 2 mL of cell
Materials Required, Not Supplied suspension with 10 mL of medium.
These materials are not included in the kit, but will be required to successfully utilize this assay: 12. Mix completely and then plate 100 μL/well (20,000 – 25,000 cells/well) onto a PDL-
 DMEM/F12 Medium (Life Technologies #11330-032) coated 96-well plate using a multi-channel pipettor or liquid handler. Throughout the
 Neurobasal Medium (Life Technologies #21103-049) seeding process, be careful not to move or agitate the plate as this may lead to
 B27 Supplement (Life Technologies #17504-044) uneven attachment.
 N2 Supplement (Life Technologies #17502-048) 13. After seeding, do not immediately transfer the plate to the incubator. Leave it in the
 GlutaMAX (Life Technologies #35050-061) hood for 15 minutes to allow the cells to settle to the bottom of the well. After 15
 Geltrex (Life Technologies #A1413201) minutes, very gently transfer the plate to a humidified incubator at 37°C with 5% CO2.
 BDNF (AB217473) Day of cell plating is designated as Day 0.
 GDNF (AB259417)
 TGF-β1 (AB50036) Δ Note: Entire thawing and plating process should not exceed 2 hours; post-thaw viability and
 PDL-Coated 96-Well Plates overall cell health will be severely impacted and lead to an unsuccessful culture.

PROCEDURE Day 1 Medium Change

Thawing and Seeding the Neurons 1. On Day 1 (24 hours after seeding), prepare fresh Day 1 Medium (see recipe below).
1. Gather the components for the Seeding Medium according to the recipe below. Note 2. Gently remove all 100 μL/well row or column wise and replace with 100 μL/well
that BDNF, GDNF, and TGF-β1 are lyophilized powders. Follow Abcam’s instructions for Day 1 Medium.
reconstitution. We recommend creating stock solutions of 10 μg/mL for BDNF, 10
μg/mL for GDNF, and 1 μg/mL for TGF-β1. Day 4 Medium Addition
2. Working in a cell culture hood (biological safety cabinet), combine all components in 1. On Day 4 (96 hours after seeding), prepare fresh Day 4 Medium (see recipe below).
an appropriately sized sterile container. For preparation of the Geltrex, add cold 2. Gently add 100 μL/well to the entire plate for a total of 200 μL/well.
DMEM/F12 directly to an aliquot of frozen Geltrex to yield a 1:10 dilution. For example,
if aliquots of Geltrex have a volume of 100 μL, add 900 μL of cold DMEM/F12. Δ Note: If only culturing for 7 days, then Supplement K can be removed from Day 4 Medium.
Immediately place this mixture at 4°C to allow the Geltrex to thaw and dissolve before
adding the appropriate amount to the Seeding Medium. Allow the Seeding Medium Day 7 and Onward Medium Changes
to equilibrate to room temperature for at least 15 minutes. Do not warm the medium in 1. Change half the medium (100 μL/well) twice weekly (on Day 7, 11, 14, 18, etc.) using
a 37°C water bath. Maintenance Medium (see recipe below).
3. Remove the cryovial from liquid nitrogen and place in a 37°C water bath. To minimize 2. The neurons mature rapidly and can be maintained viable and adherent in culture
contamination, avoid submerging the cap. Gently move the vial within the bath to under the above conditions for at least 3 weeks post-seeding.
increase the rate of thawing.

Version 1, Last updated December 13, 2023

Media Compositions

Seeding Medium Maintenance Medium

Component Stock Final 1 plate 2 plate 5 plate Component Stock Final 1 plate 2 plate 5 plate
Conc. Conc. volumes volumes volumes Conc. Conc. volumes volumes volumes
1 DMEM/F12 Medium 1X 0.5X 9.5 mL 19 mL 47.5 mL 1 DMEM/F12 Medium 1X 0.5X 9.6 mL 19.2 mL 48 mL
2 Neurobasal Medium 1X 0.5X 9.5 mL 19 mL 47.5 mL 2 Neurobasal Medium 1X 0.5X 9.6 mL 19.2 mL 48 mL
3 B27 Supplement 50X 1X 400 µL 800 µL 2 mL 3 B27 Supplement 50X 1X 400 µL 800 µL 2 mL
4 N2 Supplement 100X 1X 200 µL 400 µL 1 mL 4 N2 Supplement 100X 1X 200 µL 400 µL 1 mL
5 GlutaMAX 200 mM 0.5 mM 50 µL 100 µL 250 µL 5 GlutaMAX 200 mM 0.5 mM 50 µL 100 µL 250 µL
6 BDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL 6 BDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL
7 GDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL 7 GDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL
8 TGF-β1 1 µg/mL 1 ng/mL 20 µL 40 µL 100 µL 8 TGF-β1 1 µg/mL 1 ng/mL 20 µL 40 µL 100 µL
9 Geltrex 15 mg/mL 15 µg/mL 200 µL 400 µL 1 mL
(of 1:10) (of 1:10) (of 1:10)
10 BrainFast Motor 1000X 1X 20 µL 40 µL 100 µL
Supplement (1000x)
Technical Support
Day 1 Medium Copyright © 2023 Abcam. All Rights Reserved. The Abcam logo is a registered trademark.
Component Stock Final 1 plate 2 plate 5 plate All information / detail is correct at the time of printing.
Conc. Conc. volumes volumes volumes For all technical or commercial enquiries please go to:
1 DMEM/F12 Medium 1X 0.5X 9.5 mL 19 mL 47.5 mL
2 Neurobasal Medium 1X 0.5X 9.5 mL 19 mL 47.5 mL www.abcam.com/contactus
3 B27 Supplement 50X 1X 400 µL 800 µL 2 mL www.abcam.cn/contactus (China)
4 N2 Supplement 100X 1X 200 µL 400 µL 1 mL www.abcam.co.jp/contactus (Japan)
5 GlutaMAX 200 mM 0.5 mM 50 µL 100 µL 250 µL
6 BDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL
7 GDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL
8 TGF-β1 1 µg/mL 1 ng/mL 20 µL 40 µL 100 µL
9 BrainFast SK 1000X 1X 20 µL 40 µL 100 µL
Supplement (1000x)
10 BrainFast Motor 1000X 1X 20 µL 40 µL 100 µL
Supplement (1000x)

Day 4 Medium
Component Stock Final 1 plate 2 plate 5 plate
Conc. Conc. volumes volumes volumes
1 DMEM/F12 Medium 1X 0.5X 9.6 mL 19.2 mL 48 mL
2 Neurobasal Medium 1X 0.5X 9.6 mL 19.2 mL 48 mL
3 B27 Supplement 50X 1X 400 µL 800 µL 2 mL
4 N2 Supplement 100X 1X 200 µL 400 µL 1 mL
5 GlutaMAX 200 mM 0.5 mM 50 µL 100 µL 250 µL
6 BDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL
7 GDNF 10 µg/mL 10 ng/mL 20 µL 40 µL 100 µL
8 TGF-β1 1 µg/mL 1 ng/mL 20 µL 40 µL 100 µL
9 BrainFast D4 1000X 1X 20 µL 40 µL 100 µL
Supplement (1000x)
10 BrainFast SK 1000X 1X 20 µL 40 µL 100 µL
Supplement (1000x)

Version 1, Last updated December 13, 2023