Neural Progenitor Cells Derived from XCL-1 DCXp-GFP

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

Post-thaw: 8.0 x 10⁴ viable cells/cm² on Cell Basement Membrane-coated dishes/

Subculture: 4.0 x 10⁴ viable cells/cm² on Cell Basement Membrane-coated dishes/plates

Coat plates with Cell Basement Membrane (ATCC ACS-3035) and culture the NPCs with NPC Growth Medium (ATCC ACS-3003).

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If, upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –80°C. Storage at –80°C will result in loss of viability. Preparation of Cell Basement Membrane (ATCC ACS-3035) coated plates:

1. Thaw Cell Basement Membrane on ice or at 4°C
2. Prepare a 150 µg/ mL working concentration of Cell Basement Membrane in cold DMEM: F-12 medium
3. Add enough Cell Basement Membrane solution to cover the surface of the plate (e.g. 1 mL diluted Cell Basement Membrane/well of a 12-well plate)
4. Incubate for 1 hour at 37°C prior to use

Initiation of Cultures

1. Prepare complete NPC growth medium (ATCC ACS-3003) following the instructions in the package and pre-warm that medium as well as DMEM:F12 in a 37°C water bath for 15-30 min. If using a small volume of medium (50 mL or less), warm only the volume needed in a sterile conical tube. Avoid warming complete medium multiple times.
2. Obtain a 12-well plate with Cell Basement Membrane. Aspirate the Cell Basement Membrane medium and directly add 1.5 mL of the complete NPC Growth Medium per well. Place the plate in the incubator for 15 minutes to allow the medium to reach its normal pH (7.0-7.6). Four to five wells of a 6-well plate may be needed for each vial of cells thawed.
3. Transfer 9 mL of pre-warmed DMEM:F12 into a 15 mL conical tube for recovery of the NPCs from the frozen stock.
4. Remove cryovial of frozen cells from liquid nitrogen storage.
5. Thaw the cells by gently swirling in a 37°C water bath. To reduce the possibility of contamination, keep the cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes). Remove the cryovial from water bath when only a few ice crystals are remaining.
6. Sterilize the cryovial with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
7. Remove cells from the vial using a P1000 micropipette and transfer cells drop-wise into the 15 mL conical tube containing 9 mL DMEM:F12.
8. Centrifuge cells at 270 x g for 5 minutes at room temperature.
9. Aspirate the supernatant and discard. Gently tap the bottom of the tube to loosen the cell pellet.
10. Add 4 mL of the complete NPC Growth Medium to the tube. Gently resuspend the pellet by pipetting up and down 3 or 4 times to make a single-cell suspension.
11. Perform cell count by a Vi-Cell Analyzer or hemocytometer. Note: Don’t perform cell count by a Vi-Cell Analyzer without removal of serum-free freezing medium.
12. Seed NPCs at a seeding density of 80,000 viable cells/cm² (e.g. 0.30 x10⁶/well of a 12-well plate) onto a Cell Basement Membrane-coated plate described above.
13. Incubate the plate at 37°C with 5% CO₂ overnight.
14. Change medium at 100% media change rate (1 mL media/well) next day and every other day thereafter.
15. Monitor cell growth and passage the cells when they reach ~95% confluency

Note: Don’t passage NPCs when the cells are <85% confluency

Post thaw day 1, perform a 100% medium change and remove all cells that did not attach. Perform a 100% medium change every other day thereafter. Passage the cells cells with diluted Accutase (50% Accutase and 50% DPBS) when they reach ~95% confluence and reseed the NPCs at 40,000 viable cells/cm² on Cell Basement Membrane Gel-coated dishes/plates.

Post thaw day 1, perform a 100% medium change and remove all cells that did not attach. Perform a 100% medium change every other day thereafter. Passage the cells cells with diluted Accutase (50% Accutase and 50% DPBS) when they reach ~95% confluence and reseed the NPCs at 40,000 viable cells/cm² on CellMatrix-coated dishes/plates.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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