TITLE: Splitting p0 (6wp) to p1 (T75)
AUTHORS: Andrea Argouarch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol includes splitting outgrowth of dural cell from a 6 well plate into one T75 flask for expansion. </div></div>

[STEPS]
?. [Preparation]
Turn off UV lights and clean hood with 70% ethanol
?. [Clean Up]
Throw away biohazard materials properly     a. 10 cm dishes with glass coverslips should be thrown away in biohazard red sharp container
?. [Clean Up]
Clean surgical tools, wear waterproof lab coat and eye protection/PPE    a. Brush and clean with 409 soap water. Rinse with water, dry on kimwipe, rinse with 100% ethanol, and then dry completely with kimwipe to prevent rust or water marks.     b. Prep for next autoclaving cycle
?. [Clean Up]
Clean and sterilize hood with 70% ethanol and turn on UV
?. [Clean Up]
Update cell culture notes in lab notebook
?. [Observations]
After 3 weeks, split 6 wells of the 6 well plate (6wx6wp) into 1xT75 flask    a. Keep 6wp as backup by re-feeding the entire plate after trypsinization.
?. [Observations]
Cells will grow on the plastic and maybe on the glass coverslip itself
?. [Preparation]
Clean items with 70% ethanol and bring into hood    a. Autoclaved  # 5 Forceps     b. 10 cm dish for forceps, cell scraper, discarded coverslips, and discarded tissue     c. Sterile cell scraper    d. DPBS -/-     e. Sterile Filtered Media with PenStrep     f. Label 1xT75 flask with ID, date, and p1, add 15 mls   of media per flask    g. 0.05% Trypsin         i. Aliquot and warm before use
15 mL
?. [Culturing]
Aspirate old media
?. [Culturing]
Rinse by adding 3 mls   of DPBS per well
3 mL
?. [Culturing]
Lift coverslips with forceps to allow adequate washing under the coverslip and gently swirl
?. [Culturing]
Aspirate DPBS
?. [Culturing]
Add 2 ml   of trypsin per well and lift coverslips breifly with forceps
2 mL
?. [Culturing]
Place in incubator   for ~5 mins   until cells start to detach     a. Can also gently tap the side of the plate to detach cells from the coverslip
37 °C
?. [Culturing]
Add 2 mls   of media per well to inactivate trypsin
2 mL
?. [Culturing]
With forceps, flip over coverslip within each well    a. Can remove tissue and place in 10 cm dish to discard
?. [Culturing]
Use cell scraper to gently scrap off cells in each well attached to the coverslip
?. [Culturing]
Discard each coverslip in the 10 cm dish
?. [Culturing]
Use cell scraper to scrap off remaining cells (cells that are attached to bottom of plate)
?. [Culturing]
Check under microscope and mark areas with cells still attached with a pen
?. [Culturing]
Re-scrape cell gently if needed
?. [Culturing]
Collect cell suspension in a 50 ml conical
?. [Culturing]
Rinse wells with additional media ~6mls   to collect any remaining cells
6 mL
?. [Culturing]
Spin at 1000 rpm   for 5 mins
Centrifuge: 1000 33
?. [Culturing]
Re-feed old 6wp plate for backup, 2 mls   per well
2 mL
?. [Culturing]
Aspirate supernatant, being careful to not aspirate the pellet
?. [Culturing]
Tap the pellet to resuspend
?. [Culturing]
Add media to a final volume of 5 mls  and pipette up and down to mix
5 mL
?. [Culturing]
Add cell suspension to flask
?. [Culturing]
Place flask in incubator (5% CO2, 37oC)
37 °C
?. [Culturing]
Observe and feed the next day to remove debris, then feed every 2-3 days until 90-100% confluent