Thermo Scientific Neurobasal-A Medium User manual

Neurobasal™Medium and Neurobasal™‑A Medium
Catalog Numbers 21103049, 10888022, 12348017, and 12349015
Pub. No. MAN0007306 Rev. 2.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermosher.com/support.
Product description
Neurobasal™ Medium and Neurobasal™‑A Medium are basal media that, when supplemented with B‑27™ Supplement, meet the special cell
culture requirements of pre‑natal/embryonic and post‑natal/adult brain neuronal cells, respectively. Both Neurobasal™ Medium and
Neurobasal™‑A Medium can be used to cultivate neuronal cells from hippocampus, cortex and other regions of the brain. Both media when
supplemented with B‑27™ Supplement have demonstrated optimal viability for both long and short term maintenance of homogeneous
populations (<0.5% Glial cells) of neuronal cells without the need for an astrocyte feeder layer. Neurobasal™ Medium Minus Phenol Red
and Neurobasal™‑A Medium Minus Phenol Red are provided for receptor studies such as estrogenic receptors, downstream protein
purication studies or other processes where the presence of phenol red is undesirable.
Contents and storage
Contents Cat. No. Amount Storage Shelf life[1]
Neurobasal™Medium 21103049 500 mL
2–8°C; Protect from light 12 months
Neurobasal™Medium Minus Phenol Red 12348017 500 mL
Neurobasal™‑A Medium 10888022 500 mL
Neurobasal™-A Medium Minus Phenol Red 12349015 500 mL
[1] Shelf life duration is determined from Date of Manufacture.
Procedural guidelines
• Neurobasal™ Medium or Neurobasal™‑A Medium, when
supplemented with B‑27™ Supplement, contain anti‑oxidants
to reduce reactive oxygen damage and they do not contain
the excitatory amino acids, glutamate and aspartate, making
them amenable to the study of these neurotransmiers.
• Neurobasal™‑A Medium, when supplemented with B‑27™
Supplement, is eective for the growth of tumor cell lines of
neuronal origin.
Culture conditions
Media: Complete Neurobasal™ Medium or Neurobasal™‑A
Medium
Culture type: Adherent
Culture vessels: Multiwell plate or T‑asks
Temperature range: 36°C to 38°C
Incubator atmosphere: Humidied atmosphere of 5% CO2 in air.
Ensure proper gas exchange and minimize exposure of cultures to
light.
Prepare complete media
1. Aseptically add supplements to 100 mL Neurobasal™
Medium or Neurobasal™‑A Medium according to one of the
following conditions.
• Add 2 mL B‑27™ Supplement or other B‑27™ Supplement
variants and 0.5 mM L‑glutamine or GlutaMAX™
Supplement.
• Add 1 mL N‑2 Supplement (100X) and 0.5–
2 mM L‑glutamine or GlutaMAX™ Supplement.
• Add 1 mL G‑5 Supplement (100X) and 0.5–
2 mM L‑glutamine or GlutaMAX™ Supplement.
2. Prior to initial plating of primary hippocampal neurons,
further supplement Neurobasal™ Medium with 25 µM
(3.7 µg/mL) glutamate.
Some cell lines may require an initial aachment in
2% serum‑supplemented complete Neurobasal™ Medium.
Once supplemented, the complete Neurobasal™ Medium is
stable for up to one week when stored in the dark at 2°C to
8°C.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.

Cell culture procedure
Coat culture plates with Poly-D-Lysine
1. Dilute the Poly‑D‑Lysine solution in sterile DPBS to prepare
a 50 µg/mL working solution.
2. Coat the surface of the culture vessel (German glass or cell
culture grade plastic) with 50 µg/mL Poly‑D‑Lysine solution.
For primary neurons, use 0.15 mL/cm2 surface area.
3. Incubate the vessel for 1 hour at room temperature.
4. Remove the Poly‑D‑Lysine solution, and rinse the culture
surface 3 times with sterile distilled water.
Make sure to rinse the culture vessel thoroughly as excess
Poly‑D‑Lysine solution can be toxic to the cells.
5. Remove distilled water and leave the coated culture vessel
uncovered in the laminar hood to dry.
The culture surface will be fully dry after 2 hours.
Plates can be used immediately once dry or can be stored dry
at 4°C. For storage at 4°C, tightly wrap the vessel with
Paralm™ lm and use within one week of coating.
Culture neurons
1. Isolate primary rat neurons or thaw cryopreserved primary
rat neurons according to standard laboratory procedure or
instructions supplied with the cells; see “Recover
cryopreserved cells“.
2. Plate cells in pre‑warmed (37°C) complete
Neurobasal™‑A/B‑27™ medium (postnatal) or
Neurobasal™/B‑27™ medium (prenatal), at a suggested
density of 160 cells/mm², or another optimized density if
required.
3. Incubate the culture dish at 36°C to 38°C in a humidied
atmosphere of 5% CO2 (in air is acceptable but 9% oxygen
with 5% CO2 is preferable).
4. After 4–24 hour incubation, aspirate half of the medium and
replace with same volume of fresh medium.
Return the plate to the incubator.
5. Refeed cells every 3–4 days by removing half of the medium
and replacing it with an equal volume.
6. Refeed cells (day 3 or 4 post‑plating and every 3 days
thereafter) by removing one‑half of the medium and
replacing with an equal volume.
Medium changes for prenatal neurons should be made with
Neurobasal™/B‑27™ medium without glutamate, to reduce
glutamate toxicity in the culture. For postnatal neurons use
Neurobasal™‑A/B‑27™ medium, without glutamate,
supplemented with 10 ng/mL bFGF.
Note: Include glutamate in the medium for plating and
subsequent media changes when culturing neuroblastoma
cells.
Recover cryopreserved cells
Primary neuronal cells are extremely fragile upon recovery from
cryopreservation.
IMPORTANT! Do not centrifuge cells.
Primary neuronal cells will adhere to bare plastic and glassware;
to maximize cell recovery and yield we recommend pre‑rinsing all
plastic and glassware with complete Neurobasal™/B‑27™ medium
before use.
1. Prepare Poly‑D‑Lysine coated sterile culture vessels ahead of
time (see “Coat culture plates with Poly‑D‑Lysine“).
2. Rapidly thaw (<1 minute) frozen vial of cells in a 37°C water
bath.
Remove vial from water bath just before the last trace of ice
has melted.
3. Rinse a pipee tip with complete medium and very gently
transfer the cells from the cryovial to a prerinsed 15‑mL
conical tube.
4. Rinse the cryovial with 1 mL of pre‑warmed complete
Neurobasal™/B‑27™ medium, and transfer the rinse to the 15‑
mL tube containing the cells at a rate of one drop per second.
Mix by gentle swirling after each drop.
5. Dropwise add 2 mL of complete Neurobasal™/B‑27™ medium
to the tube (for a total suspension volume of 4 mL).
Mix by gentle swirling after each drop.
6. Determine viable cell density using a Countess™ II
Automated Cell Counter.
7. Plate ~1 × 105 cells per well in Poly‑D‑Lysine coated 48‑well
plate or an 8‑chambered slide. Bring the cell suspension
volume to 500 µL per well by adding complete
Neurobasal™/B‑27™ medium.
8. Incubate the cells at 37°C in a humidied atmosphere of
5% CO2 in air (9% oxygen with 5% CO2 is preferable).
9. See “Culture neurons“ step 4–step 6.
2 Neurobasal
™
Medium and Neurobasal
™
‑A Medium User Guide

Related products
Unless otherwise indicated, all materials are available through thermosher.com.
Item Source
B-27™Supplement (50X) 17504
B-27™Supplement (50X) minus antioxidants 10889
B-27™Supplement (50X) minus vitamin A 12587
G‑5 Supplement (100X) 17503
N‑2 Supplement (100X) 17502
GlutaMAX™Supplement 35050
L‑Glutamine, 200 mM (100X) 25030
Poly-D-Lysine A3890401
Primary Rat Cortex Neurons A10840
Primary Rat Hippocampus Neurons A10841
Penicillin-Streptomycin 15070
bFGF Recombinant Human Protein 13256
2‑Mercaptoethanol (1000X) 21985
DPBS, no calcium, no magnesium 14190
Countess™II Automated Cell Counter AMQAX1000
Explanation of symbols
Symbol Description Symbol Description Symbol Description
Manufacturer Catalog number Batch code
Use by Temperature limitation Keep away from light
Sterilized using aseptic
processing techniques Consult instructions for use Caution, consult accompanying documents
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and
Conditions of Sale found on Life Technologies' website at www.thermosher.com/us/en/home/global/terms-and-conditions.html. If you
have any questions, please contact Life Technologies at www.thermosher.com/support.
Manufacturer: Life Technologies Corporation | 3175 Staley Road | Grand Island, NY 14072
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. B-27™is a
trademark of Southern Illinois University.
Neurobasal
™
Medium and Neurobasal
™
‑A Medium User Guide 3

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14 August 2018