Cell Culture Basics by Shannon Patrick https://padlet.com/spatri11/uoub7okwsrf5g0au en-us 2022-02-01 16:20:41 UTC 2022-02-01 16:54:20 UTC hello@padlet.com spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023721560 1. Sterilize the hood and needed supplies]]> 2022-02-01 16:26:51 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023721560 spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023722760 2. Examine culture for signs of contamination]]> 2022-02-01 16:27:20 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023722760 Adherent Cells spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023726927 1. Remove the spent medium from the flask using a sterile pipette.
2. Rinse the cells with a balanced salt solution, such as DPPS.
3. After rinsing the cells, remove the salt solution.
4. Each time you aspirate liquid off the cells, place the next solution on quickly.
5. Add your cell dissociation reagent to remove cells from the plate. Use just enough solution to cover the cell sheet.
6. Tap gently on the plate to help the cells detach. Use a microscope to confirm the cells have released from the flask.
(Complete dissociation is necessary).
7. Can manually break up lingering clumps by repeatedly pipetting warmed medium over them.
8. If using trypsin: the collection medium will need serum or  a trypsin inhibitor to inactivate the trypsin. If using TrypLE: inactivation is achieved by dilution alone. No serum is needed.
9. Transfer the cells to a conical tube and centrifuge to remove any residual dissociation reagent.
10. After centrifugation, there should be a well-formed pellet.

]]> 2022-02-01 16:29:15 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023726927 Adherent cells spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023728701 Using a salt solution with calcium and magnesium may inhibit your cell dissociation reagent.

Not using a gentle dissociation reagent, such as TrypLE Express, may cause damage to your cells during dissociation.

Do not leave the dissociation reagent on too long, especially if you're using a reagent other than TrypLE.

Not achieving a single-cell suspension will give an inaccurate cell count.

Leaving try blue on too long will cause live cells to turn blue, giving an inaccurate live cell count.

Capping unvented caps too tightly will preventing adequate gas exchange.]]> 2022-02-01 16:30:04 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023728701 spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023745945 11. Remove the medium from the centrifuge tube with a pipette and discard the medium into a waste container. Try not to disturb the pellet.
12. Resuspend the pellet with warm complete growth medium. Gentle pipetting will disperse the cells to ensure a homogeneous solution of single cells.
13. Remove a small sample for cell counting.
14. Trypan blue is used when counting cells to indicate the ratio of live to dead cells. Stain turns dead cells blue, and healthy cells remain white or colorless.
15. Using the microscope, count the total number of cells and the number of dead or blue cells.
16. Based on your cell count, determine how much additional fresh medium to add for optimal seeding density.
17. Add the required medium, mix the cells gently, and pipette the solution into fresh flasks.
18. Cap the vented caps on your flask tightly. If the caps are not vented, cap loosely. 
19. Use a north, south, east, west motion to evenly distribute the cells and transport the flasks to the incubator.

]]> 2022-02-01 16:37:15 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023745945 Suspension Cells spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023754985 Not staying within the minimum and maximum volumes when adding new medium, which will diminish optimal air exchange and shaking flow.

Capping the flasks inappropriately, either vented or not. 

Not spinning the cells or replacing medium in a timely manner.

 
  
]]> 2022-02-01 16:41:03 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023754985 What Could Go Wrong? spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023755513 2022-02-01 16:41:16 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023755513 Suspension Cells spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023760589 1. Remove a small sample from the cell culture flask for counting.
2. You'll follow the same counting procedure for adherent cells, using trypan blue and either a hemocytometer or the Countess automated cell counter.
3. Based on your cell count, add additional fresh medium to the flasks. (Stay within the minimum and maximum volumes.)
4. You may need to split the culture into multiple flasks.
5. Cap the flasks appropriately, depending upon whether they are vented or not, and return them to the incubator.
6. Every three weeks, gently spin the cells, remove the medium, and replace with completely new medium. (This eliminates cell debris and metabolic waste).
 
  
]]> 2022-02-01 16:43:20 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023760589 Passaging Cells Steps spatri11 https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023782406 2022-02-01 16:52:24 UTC https://padlet.com/spatri11/uoub7okwsrf5g0au/wish/2023782406