B-27 Plus Neuronal Culture System

Transcription

1 USER GUIDE B-27 Plus Neuronal Culture System Catalog Number A Pub. No. MAN Rev. 1.0 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 thermofisher.com/support. Product description The Gibco B-27 Plus Neuronal Culture System is comprised of B-27 Plus Supplement (50X) and Neurobasal Plus Medium. This system represents an evolution of the neuronal cell culture products, B-27 Supplement and Neurobasal Medium, that is designed to provide maximum in vitro survival of primary rodent and human stem cell-derived neurons. Contents and storage Table 1 B-27 Plus Neuronal Culture System, Cat. No. A Contents Cat. No. Amount Storage Shelf life [1] Neurobasal Plus Medium A ml 2 C to 8 C; Protect from light. B-27 Plus Supplement (50X) A ml 20 C to 5 C; Protect from light. 12 months [1] Shelf-Life duration is determined from Date of Manufacture. Use Maintenance of primary rat, mouse and human PSC-derived and fetal-derived neurons. Differentiation of human PSC-derived and fetal-derived Neural Stem Cells (NSC) to neurons. Prepare complete Neurobasal Plus Medium (Maintenance) 1. Aseptically add GlutaMAX I Supplement to 0.5 mm concentration (2.5 ml/l) to the medium before use. Note: Unlike Neurobasal Medium, Neurobasal Plus Medium contains 0.5 mm GlutaMAX I Supplement; however we have tested supplementing additional GlutaMAX I Supplement up to 2 mm final concentration with no detrimental effects on neuronal survival. 2. Thaw B-27 Plus Supplement (50X) overnight at 4 C. Aseptically add 2% B-27 Plus Supplement (50X) (20 ml/l) to the Neurobasal Plus Medium before use. Note: Remaining B-27 Plus Supplement (50X) may be aliquoted into working volumes and stored at 20 C to 5 C. Thaw aliquots as needed. Do not freeze-thaw B-27 Plus Supplement (50X) more than twice. Once thawed do not leave thawed supplement at 4 C for more than two weeks. 3. Pre-warm complete Neurobasal Plus Medium at 37 C in a water bath before using. Cell culture procedure The following procedure has been tested on freshly isolated 18-day gestation rat hippocampal and cortical neurons, Gibco Primary Rat Cortex Neurons, Gibco Primary Rat Hippocampus Neurons, Gibco Primary Mouse Cortex Neurons, Gibco Primary Mouse Hippocampus Neurons and human PSC-derived neurons. Coat culture plates with poly-d-lysine 1. Prepare a 2-mg/mL poly-d-lysine stock solution in distilled water. 2. Dilute the poly-d-lysine stock solution 1:40 in D-PBS to prepare a 50 μg/ml working solution (i.e., 125 μl of poly-dlysine stock solution into 5 ml of DPBS, calcium, magnesium). 3. Coat the surface of the culture vessel with the working solution of poly-d-lysine (150 μl/ cm 2, i.e., 50 μl per well for a 96-well plate). 4. Incubate the culture vessel at room temperature for 1 hour. For Research Use Only. Not for use in diagnostic procedures.

2 5. Remove the poly-d-lysine solution and rinse 3 times with distilled water. Make sure to rinse the culture vessel thoroughly, because excess poly-d-lysine can be toxic to the cells. 6. Leave the coated vessels uncovered in the laminar hood until the wells have completely dried. You may use the dry plates immediately or store them at 4 C, wrapped tightly with Parafilm wrapper, for up to one week. Culture neurons 1. Isolate primary neurons according to standard laboratory procedures or thaw cryopreserved neurons according to instructions supplied with the cells. See Recover and culture cryopreserved neurons. 2. Plate cells on poly-d-lysine coated vessel using pre-warmed (37 C) complete Neurobasal Plus Medium (prepared as described above) at a suggested density of approximately viable cells/cm 2, or another optimized density if required. Note: For rat and mouse hippocampal neurons, use the complete Neurobasal Plus Medium supplemented with 25 μm L-Glutamate. 3. Incubate the culture dish at 36 C to 38 C in a humidified atmosphere of 5% CO Non-hippocampal cultures: Four days after plating, feed the cultures by aspirating half of the medium from each well and replacing with same volume of fresh medium. Repeat every three days thereafter. Hippocampal cultures: Three days after plating feed the cultures by aspirating half of the medium from each well and replacing with same volume of fresh medium without L- Glutamate. Recover and culture cryopreserved neurons procedure Procedural guidelines 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 medium before use. Do not vortex or centrifuge cells at any time during this procedure as cells are extremely fragile upon recovery from cryopreservation. Thaw one vial of neuronal cells at a time. Transfer cryovial from liquid nitrogen storage to 37 C water bath minimizing handling time. A small amount of liquid nitrogen in an ice bucket can be used to transport the vials from liquid nitrogen to the water bath. Recover and culture cryopreserved neurons 1. Rinse a sterile 15-mL conical culture tube with complete Neurobasal Plus Medium and leave in the hood prior to thawing cells. 2. If removing vial from liquid nitrogen storage, twist cap slightly to release pressure and then re-tighten cap. 3. Rapidly thaw (<2 minutes) frozen vial by gently swirling in a 37 C water bath. Remove from water bath when only one tiny ice crystal is left; the vial should still be cold to the touch. 4. Transfer the vial into the hood and disinfect with 70% isopropyl alcohol. Collect the liquid to the bottom of the vial by gently tapping the vial on the hood s surface. 5. Use a pre-rinsed 1-mL pipette tip to very gently transfer the cells to the pre-rinsed 15-mL conical tube. 6. Rinse the cryovial with 1 ml of pre-warmed complete Neurobasal Plus Medium and extremely slowly add to the cells in the 15-mL tube at the rate of one drop per second. Mix by gentle swirling after each drop. Do not add the full amount of media to the tube at once. This may lead to decreased cell viability due to osmotic shock. 7. Slowly (dropwise) add an additional 2 ml of pre-warmed complete Neurobasal Plus Medium to the tube (for a total suspension volume of 4 ml). Mix the suspension very gently with 1-mL pipette without creating any air bubbles. 8. Add 10 μl of cell suspension to a microcentrifuge tube containing 10 μl of 0.4% Trypan blue, using a pre-rinsed tip. Mix only by gently tapping the tube. Determine the viable cell density using a manual (i.e., hemocytometer) counting method. The viability of thawed cells should be >50%. 9. Plate ~ viable cells (or desired cell density) per well in a poly-d-lysine coated 96-well plate. See Coat culture plates with poly-d-lysine. 10. Dilute cell suspension to 200 μl per well by adding prewarmed complete Neurobasal Plus Medium first followed by cell suspension. 11. Incubate the culture plate at 36 C to 38 C in a humidified atmosphere of 5% CO After 4 24 hours of incubation, aspirate half of the medium and replace with same volume of fresh medium. Return the plate to the incubator. Repeat every three days thereafter. 2 B-27 Plus Neuronal Culture System User Guide

3 Differentiate neural stem cells into neurons B-27 Plus Supplement and Neurobasal Plus Medium can be used to replace B-27 Supplement and Neurobasal Medium for the differentiation of human pluripotent stem cells (PSCs) and fetal-derived neural stem cells (NSCs) into neurons. Once differentiated, continue to maintain in complete Neurobasal Plus Medium to enhance the long-term survival of differentiated neurons, as described in Culture neurons. Refer to the product manual of CultureOne Supplement (A , Pub. No. MAN ) for the complete medium preparation and cell plating procedures. Human PSC-derived NSCs are recommended to be plated at cells/cm 2 when B-27 Plus Supplement and Neurobasal Plus Medium are used with CultureOne Supplement to eliminate cell cluster formation during neuronal differentiation. The plating density of NSCs may need to be optimized depending on human PSC line used. Human fetal-derived NSCs are recommended to be plated at cells/cm 2. CultureOne Supplement is not required in the differentiation of human fetal derived NSCs into neurons. Note: B-27 Plus Neuronal Culture System is not recommended for NSC expansion. StemPro NSC SFM (Cat. No. A ) is recommended for growth and expansion of hnscs and Neural Expansion Medium is recommended for NSCs induced using Neural Induction Medium (Cat. No. A ); (Pub. No. MAN ). Characterize neurons 1. At the end of neuronal culture, cells can be immuno-stained to characterize the neuronal population. Remove the culture medium and gently rinse the cells without dislodging them twice with DPBS, calcium, magnesium. 3. Rinse the cells three times with DPBS, calcium, magnesium. 4. Permeabilize the cells with 0.3% Triton X-100 surfactant (diluted in DPBS, calcium, magnesium) for 5 minutes at room temperature. 5. Rinse the cells three times with DPBS, calcium, magnesium. 6. Incubate cells in 5% goat serum diluted in DPBS, calcium, magnesium+ for 60 minutes at room temperature. 7. Remove the blocking solution and incubate the cells overnight with the primary antibody (mouse anti-huc/hud at 5 μg/ml and/or rabbit anti-map2 at 1:200 diluted in 5% goat serum at 4 C. Ensure that the cell surfaces are covered uniformly with the antibody solution. Note: HuC/HuD is an RNA-binding protein and binds specifically to antigens present exclusively in neuronal cell bodies. 8. Wash the cells three times for 5 minutes with DPBS, calcium, magnesium. 9. Incubate the cells with secondary antibody (Alexa Fluor 594 goat-anti rabbit (H+L) at 10 μg/ml and/or Alexa Fluor 488 goat-anti mouse (H+L) at 10 μg/ml) diluted in 5% goat serum solution for 60 minutes at room temperature. 10. Wash the cells three times with D-PBS containing DPBS, calcium, magnesium. In the last wash, counter stain the cells with DAPI solution (3 ng/ml) for 10 minutes. 11. Rinse the cells with D-PBS and observe the cells under the microscope using filters for FITC, Cy5, and DAPI. 2. Fix the cells with 4% formaldehyde solution at room temperature for 15 minutes. Examples of primary mouse and rat neuronal cultures Phase contrast image of primary mouse cortical neurons cultured at day 21 in B-27 Plus Neuronal Culture System. 2 Primary Rat cortical neurons maintained in B-27 Plus Neuronal Culture System for 35 days and stained with neuronal markers HuC/HuD (red), MAP2 (green). Cell nuclei were counter stained with DAPI (blue). B-27 Plus Neuronal Culture System User Guide 3

4 Troubleshooting Observation Possible cause Recommended action Primary rat and mouse neuronal cultures have undesired number of glial cells The presence of glial cells can be due to mixed cultures which depend on age of animals at the time of neurons isolation, isolation technique and/or initial plating density. Include CultureOne Supplement at 1X concentration at the time of plating cells to eliminate glial cells. Refer to CultureOne Supplement (A ) protocol. Delay the addition of CultureOne Supplement to days 2, 4, 6, or 8, to achieve desired levels of glial cells. There are not many cells attached Uneven substrate (PDL) coating Check if the substrate coating is even. Primary rat and mouse neuronal cultures show clumped morphology Too few cells plated Forgot to coat plate with PDL Problem with substrate coating Use freshly coated plate. Increase the cell plating density. Remember to use PDL coated plates (see protocol). Check if the substrate coating is even. Differentiating neurons form cell clumps NSCs plating density might be too high If higher NSC plating density is required for your experiment, the concentration of CultureOne Supplement (100X) can be increased to 2 4X in the final Neuronal Differentiation Medium without toxicity to neurons. Related products Product Catalog No. GlutaMAX I Supplement (100X), liquid CultureOne Supplement A Primary Rat Cortex Neurons: viable cells/vial, viable cells/vial A , A Primary Rat Hippocampus Neurons: viable cells/vial Primary Mouse Cortex Neurons : viable cells/vial, viable cells/vial Primary Mouse Hippocampus Neurons: viable cells/vial StemPro Neural Stem Cells A10841 A15585, A15586 A15587 A15654 DPBS, calcium, magnesium DPBS, no calcium, no magnesium Countess II Automated Cell Counter AMQAX1000 Trypan Blue Stain Image-iT Fixative Solution (4% formaldehyde, methanol-free) FB002 Goat Serum HuC/HuD Monoclonal Antibody A MAP2 Polyclonal Antibody PA Goat anti-mouse IgG (H+L) Highly cross-adsorbed Secondary Antibody, Alexa Fluor 488 A Goat anti-mouse IgG (H+L) Highly cross-adsorbed Secondary Antibody, Alexa Fluor 594 A Triton X-100 HFH100 Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at If you have any questions, please contact Life Technologies at 4 B-27 Plus Neuronal Culture System User Guide

5 Manufacturer: Life Technologies Corporation 3175 Staley Road Grand Island, NY The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES 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 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. thermofisher.com/support thermofisher.com/askaquestion thermofisher.com 13 September 2017