Splitting Cells Protocol

Vocabulary flashcards covering procedural details, measurements, and key parameters for the cell splitting protocol.

Sterilization Solution

$70\%$ alcohol used to sterilize the hood and all contents before starting and prior to placement inside.

Cell Confluency Threshold

The required coverage level of cells before splitting, which should be at $70\%$ or greater.

Media and PBS Warming Conditions

Pre-warmed at $37\,^\circ\text{C}$ for $15\text{-}20\,\text{min}$ prior to use.

Trypsin Warming Condition

Warmed at room temperature before being placed into the hood.

Trypsin Volume (Step 9)

$700\,\mu\text{L}\text{-}1\,\text{mL}$ of Trypsin added and distributed well; leaving it too long will kill cells.

Trypsin Incubation Parameters

Incubation in the incubator for a maximum of $3\,\text{min}$ using a timer, gently knocking flask if cells are not detached.

Trypsin Deactivation Step

Addition of $5\text{-}7\,\text{mL}$ of Media to cells to deactivate trypsin.

Cell Resuspension (Step 13)

Resuspending cells $4\text{-}5\times$ with media using a pipette; extra media without full nutrients can be used.

Centrifugation Settings

Centrifuge @ $1250\,\text{RPM}$ for $5\,\text{min}$.

Media Volume for $75\,\text{cm}^2$ Flasks

$19\,\text{mL}$ ($20\,\text{mL}$ total) of Media added to new flasks while centrifuging.

Media Volume for $25\,\text{cm}^2$ Plates

$5\text{-}7\,\text{mL}$ of media per plate.

Media Volume for $100\,\text{mm}^2$ Plates

$10\,\text{mL}$ total per plate.

Supernatant Discard

Discarding liquid supernatant from the $50\,\text{mL}$ or $15\,\text{mL}$ Falcon Tube after centrifugation.

Pellet Resuspension Volume

$1\text{-}3\,\text{mL}$ of Media used to resuspend cells, depending on pellet size.

Trypan Blue Cell Counting Ratio

A $1:1$ cell suspension ratio created by mixing $20\,\mu\text{L}$ of dye with $20\,\mu\text{L}$ of cells in an eppendorf tube and pipetting well.

Cell Counter Loading Volume

$10\,\mu\text{L}$ added from the eppendorf tube onto each side of the cell counter / each end of the slide.

Target Cell Seeding Amount

$2 \times 10^6\,\text{cells}$ added to each new flask (each containing $20\,\text{mL}$ total volume).

Trypsin Re-Label Reminder

Highlighted instruction at the bottom of the protocol emphasizing: 'Make Sure to Re-Label Trypsin!'.