INSTRUCTIONS FOR THAWING AND PLATING OF CELLS
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Upon receiving shipment of the cell vials, promptly remove the vials from the dry ice shipping container and immediately transfer to storage under liquid nitrogen, where cells should remain until ready for thawing.
The accompanying growth factor can be kept in the freezer or refrigerator until ready for use and the attachment factor refrigerated.
Review your thawing and plating process before initiation and move quickly and with purpose when working with these cells. Cells are negatively affected by temperature fluctuations, so pay attention to the efficiency with which you perform your protocol.
Materials Required for Thawing and Plating Cells:
· Vial of cryopreserved cells, kept properly stored under liquid N2 until the moment this protocol is initiated
· Cell Systems growth medium (appropriately supplemented with 5mLCultureBoostTM or 8mL RocketFuelTM)
· 1 vial of .25ml CultureBoost™ for resuspending cells (included with order)
· Attachment Factor™, 5mL per T-75 culture flask
· Suitable culture vessel, e.g. T-75 culture flask
· Cell culture incubator (37oC / 5% CO2 / 95% relative humidity)
· 37oC water bath
· Swinging bucket centrifuge capable of maintaining an internal temperature of 4oC
· 70-95% ethanol in spray bottle for disinfection of work surfaces and materials
· 15 mL conical tube, plastic bulb-top transfer pipette and 5&10ml serological pipettes
· Sterile lab gloves
Preparation for Thawing and Plating Cells:
1. Prewarm 14mL supplemented growth medium in a 15mL conical tube. To avoid possible contamination, make sure that
the water level never reaches the level of the tube’s cap.
2. Warm the Attachment Factor™ and supplemented growth medium to 37oC in the warm water bath.
3. Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to working under the hood.
4. Prepare your flask(s):
· Pipette Attachment Factor™ onto growth surface of flask ensuring full coverage (5mL per T-75 flask); wait ~30 seconds or more and then remove and discard
· When preparing multiple flasks, you can use the same Attachment Factor™ sequentially for up to 4 flasks.
· Add your desired volume of warm (37oC ) supplemented growth medium to the T-75 flask. Aim for a final volume of 25mL in the flask by adding 23mL at this step.
· Add Place prepared flask(s) into 37oC incubator to maintain temperature.
5. Maintain the bottle of growth medium at 37 oC for later use.
Procedure for Thawing and Plating Cells:
1. Careful observation is required here. Thaw the cryopreserved vial for less than 1 minute in your 37oC water bath. Make certain that the water does not reach the cap of the vial.
· The goal is to maintain the temperature of the cells in the vial at <0C until the moment they are placed into the 37oC medium in the culture flask.
2. As soon as half of the vial has thawed, immediately disinfect the outside of the vial with the ethanol solution and move under the hood.
3. Using your bulb top transfer pipette, carefully remove the entire volume of cells from the vial and add to the pre-warmed medium in the conical tube.
· For best results after transferring the cells, wash the inside of the vial with some of the medium from the conical tube to get as many cells out as possible.
4. Perform a cell count and check viability to determine how many cells to add to your flask(s) later. Take 10-20µL for the cell count. Cap the conical tube and place in cooled swing bucket centrifuge. Centrifuge at 500-900 g and 4C for 5-10 min.
5. Just before centrifugation is complete, move the prepared flask(s) from the incubator to under the hood.
6. Carefully remove medium supernatant from the tube by aspirating or decanting (pouring), ensuring that you do not disturb the cell pellet at the bottom of the tube.
7. Pipette 200uL of CultureBoost™ onto the cell pellet and lightly flick with your finger to loosen the pellet and gently mix the CultureBoost™ with the cells. Allow the mixture to sit for no more than 10 seconds.
8. Re-suspend the cells in the appropriate amount of warm (37oC ) growth medium using a serological pipette (e.g. 2mL).
9. Perform a second cell count and check cell viability for an accurate assessment prior to your experiment.
10. Transfer the entire volume of cell suspension into your prepared flask(s) and place into 37oC CO2 incubator.