A | B | C | |
Item | Manufacturer | Catalog Number | |
KnockOut DMEM | ThermoFisher Scientific | 10829018 | |
Growth Factor Reduced Matrigel | Corning | 356231 | |
mTeSR™ Plus and supplement | Stemcell Technologies | 1000276 | |
ReLeSR™ | Stemcell Technologies | 05872 | |
Gibco™ DPBS, no Ca, no Mg | ThermoFisher Scientific | 14-190-235 | |
Gibco™ DPBS, Ca, Mg | ThermoFisher Scientific | 14-040-117 | |
Accutase | Stemcell Technologies | 07920 | |
DMEM/F-12, HEPES | ThermoFisher Scientific | 11330032 | |
N-2 Supplement | ThermoFisher Scientific | 17502048 | |
MEM Non-Essential Amino Acids Solution (NEAA) | ThermoFisher Scientific | 11140050 | |
GlutaMAX Supplement | ThermoFisher Scientific | 35050061 | |
Culture One Supplement | ThermoFisher Scientific | A3320201 | |
B-27 Supplement, serum free | ThermoFisher Scientific | 17504044 | |
y-Secretase Inhibitor XXI, Compound E | Millipore Sigma | 565790 | |
Poly-D-Lysine (PDL) | ThermoFisher Scientific | A3890401 | |
Laminin Mouse Protein, Natural | ThermoFisher Scientific | 23017015 | |
Doxycycline Hyclate (reconstituted in water) | Millipore Sigma | D3447 | |
5-Bromo-2ʹ-deoxyuridine (BrdU) | Millipore Sigma | B9285 | |
ROCK1 Inhibitor (Y-27632 2HCl) | Selleckchem | S1049 | |
Neurobasal | Life Technologies | 21103-049 | |
HyClone Characterized Fetal Bovine Serum (FBS) | Cytiva | SH30071.03HI | |
Recombinant Human BDNF | Peprotech | 450-10 | |
Recombinant Human GDNF | Peprotech | 450-02 | |
Recombinant Human NT3 | Peprotech | 450-03 | |
Aphidicolin | Millipore Sigma | 89458 |
A | B | C | |
Equipment | Manufacturer | Catalog Number | |
Falcon® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate with Lid | Corning | 353219 | |
Eppendorf® Centrifuge 5810/5810R | Millipore Sigma | EP022628168 | |
Invitrogen Countess™ II automated cell counter | ThermoFisher Scientific | AMQAX1000 |
A | B | |
DMEM/F12 | 97 mL | |
N-2 | 1 mL | |
NEAA | 1 mL | |
GlutaMAX | 1 mL |
A | B | C | D | E | |
Cell culture vessel | Surface area (cm2) | Media volume per well | Day 3 Seeding density for ICC applications | Day 3 Seeding density for protein, RNA, DNA extraction | |
96-well plate | 0.32 | 150 µL | 20-25k cells | - | |
24-well plate | 1.9 | 500 µL | 80-100k cells | - | |
12-well plate | 3.5 | 1 mL | 2 x 105 cells | - | |
6-well plate | 9.6 | 2 mL | 0.5 x 106 cells | 1 x 106 cells | |
10-cm dish | 56.7 | 10 mL | - | 5 x 106 cells | |
15-cm dish | 145 | 30 mL | - | 15 x 106 cells |
A | B | C | D | |
Factor | Final Concentration | Stock | Dilution | |
ROCK1 inhibitor | 10 μM | 10 mM | 1:1,000 | |
Doxycycline | 2 μg/mL | 2 mg/mL | 1:1,000 | |
Compound E | 0.2 μM | 2 mM | 1:10,000 |
A | B | C | D | |
Cell culture vessel | Matrigel, PDL, and laminin in KO DMEM volume per well | DPBS volume for washes per well | Accutase® volume per well | |
96-well plate | 120 µL | 150 µL | 50 µL | |
24-well plate | 250 µL | 400 µL | 125 µL | |
12-well plate | 500 µL | 1 mL | 250 µL | |
6-well plate | 1 mL | 2 mL | 500 µL | |
10-cm dish | 4 mL | 6 mL | 3 mL | |
15-cm dish | 12 mL | 18 mL | 9 mL |
A | B | C | D | |
Cell culture vessel | Matrigel, PDL, and laminin in KO DMEM volume per well | DPBS volume for washes per well | Accutase® volume per well | |
96-well plate | 120 µL | 150 µL | 50 µL | |
24-well plate | 250 µL | 400 µL | 125 µL | |
12-well plate | 500 µL | 1 mL | 250 µL | |
6-well plate | 1 mL | 2 mL | 500 µL | |
10-cm dish | 4 mL | 6 mL | 3 mL | |
15-cm dish | 12 mL | 18 mL | 9 mL |
A | B | C | D | E | |
Cell culture vessel | Surface area (cm2) | Media volume per well | Day 3 Seeding density for ICC applications | Day 3 Seeding density for protein, RNA, DNA extraction | |
96-well plate | 0.32 | 150 µL | 20-25k cells | - | |
24-well plate | 1.9 | 500 µL | 80-100k cells | - | |
12-well plate | 3.5 | 1 mL | 2 x 105 cells | - | |
6-well plate | 9.6 | 2 mL | 0.5 x 106 cells | 1 x 106 cells | |
10-cm dish | 56.7 | 10 mL | - | 5 x 106 cells | |
15-cm dish | 145 | 30 mL | - | 15 x 106 cells |
A | B | C | D | |
Factor | Final Concentration | Stock | Dilution | |
ROCK1 inhibitor | 10 μM | 10 mM | 1:1,000 | |
Doxycycline | 2 μg/mL | 2 mg/mL | 1:1,000 | |
Compound E | 0.2 μM | 2 mM | 1:10,000 | |
Laminin | 1 μg/mL | 1 mg/mL | 1:1,000 | |
BrdU | 40 μM | 40 mM | 1:1,000 |
A | B | C | D | |
Factor | Final Concentration | Stock | Dilution | |
B-27 Supplement | 50x | 01:50 | ||
Culture One Supplement | 100x | 1:100 | ||
Laminin | 1 μg/mL | 1 mg/mL | 1:1,000 | |
BDNF | 20 ng/mL | 20 μg/mL | 1:1,000 | |
GDNF | 20 ng/mL | 20 μg/mL | 1:1,000 | |
NT3 | 20 ng/mL | 20 μg/mL | 1:1,000 |
A | B | C | D | |
Factor | Final Concentration | Stock | Dilution | |
B-27 Supplement | 50x | 1:50 | ||
Culture One Supplement | 100x | 1:100 | ||
Laminin | 500 ng/mL | 1 mg/mL | 1:1,000 | |
BDNF | 20 ng/mL | 20 μg/mL | 1:500 | |
GDNF | 20 ng/mL | 20 μg/mL | 1:500 | |
NT3 | 20 ng/mL | 20 μg/mL | 1:500 |
A | B | |
Neurobasal | 97 mL | |
N-2 | 1 mL | |
NEAA | 1 mL | |
GlutaMAX | 1 mL |
A | B | C | D | |
Factor | Final Concentration | Stock | Dilution | |
B-27 Supplement | 50x | 1:50 | ||
Culture One Supplement | 100x | 1:100 | ||
Laminin | 500 ng/mL | 1 mg/mL | 1:1,000 | |
BDNF | 20 ng/mL | 20 μg/mL | 1:500 | |
GDNF | 20 ng/mL | 20 μg/mL | 1:500 | |
NT3 | 20 ng/mL | 20 μg/mL | 1:500 |