TITLE: Freezing/Cell Banking
AUTHORS: Andrea Argouarch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol includes banking dural cells into cryovials for long term storage in liquid nitrogen. </div></div>

[STEPS]
?. [Observations]
At 100% confluency, take pictures of cell morphology, freeze down for banking (~0.5 Million per vial), collect remaining cells in flask for gDNA, and seed cells into 1xT25 for karyotyping (~30,000 cells, 60 ul)
?. [Preparation]
Turn off UV lights and clean hood with 70% ethanol
?. [Preparation]
Labeling cryovials     a. Create labels using Microsoft Word, following the formatting instructions     b. Print out estimated number of labels for the both flasks (~15) with ID, date, p2, and ~0.5 mill cells.    c. Arrange cryovials in 96, 1.5ml tube holder with spaces in between each vial    d. Spray label sheets thoroughly with 70% ethanol, dry, and bring into hood to adhere onto each vial    e. Label top of each vial with ID    f. Unscrew cap in order to easily lift up when pipetting in cell suspension
?. [Preparation]
Clean items with 70% ethanol and bring into hood    a. DPBS -/-    b. Sterile Filtered Media     c. 0.05% Trypsin        i. Aliquot to and warm before use    d. Freezing media (made fresh)        i. 100% DMSO to 10%         ii. 90% Mediae. Incyto disposable hemocytometersf. Trypan blue, add 40 ul   into a 1.5 ml tubeg. T25 labeled to send to Cell Line Genetics for karyotyping (KT)h. 15 ml conical with 10 mls   of media to dilute KT cellsi. 15 ml conical to collect cells for gDNA
40 µl
10 mL
?. [Culturing]
Take 3-4 BF images of morphology. Save TIFF under ID and passage number
?. [Culturing]
Aspirate old media
?. [Culturing]
Rinse by adding 5 mls   of DPBS per flask and gently swirling
5 mL
?. [Culturing]
Aspirate DPBS
?. [Culturing]
Add 3 ml   of trypsin per flask and place in incubator   for 3 mins   until cells are  detached     a. Can also gently tap the side of the flask to detach cells, check under microscope to confirm
3 mL
37 °C
?. [Culturing]
Add 9 mls   of fibroblast media per flask to inactivate trypsin
9 mL
?. [Culturing]
Collect cell suspension in a 50 ml conical
?. [Culturing]
Rinse flask with 12 mls   of PBS to collect remaining cells, place in 15ml conical for gDNA
12 mL
?. [Culturing]
Measure final cell suspension volume with serological and mix
?. [Culturing]
Dilute 10 ul   of cell suspension into trypan blue, tap to mix
10 µl
?. [Culturing]
Load 10 ul   of trypan blue and cell dilution into the hemocytometer
10 µl
?. [Culturing]
Count non blue cells in 4 grind corners during spin and then calculate total cells and resuspension volume for 0.5E6 cells per ml
?. [Culturing]
Total cells for 2 flasks = (Cell Count/4 grinds) x 5 dilution factor x 10,000 x ~25mls suspension volume     a. Resuspension volume for 0.5E6 cells per ml = Number of vials
?. [Culturing]
Spin both conicals at 1000 rpm  for 5 mins   during counting
Centrifuge: 1000 33
?. [Culturing]
Aspirate supernatant for both pellets (freezing and gDNA), being careful to not aspirate the pellet
?. [Culturing]
Tap the pellet to resuspend for freezing
?. [Culturing]
Store gDNA pellet at -80C   until mycoplasma assay
-80 °C
?. [Culturing]
Add freezing media dropwise to cell pellet and add 1 ml   per cryovial. Spin KT cell pellet during this time.
1 mL
?. [Culturing]
Secure each cryovial cap tightly and place in Mr. Frosty
?. [Culturing]
Place Mr. Frosty in -80oC   for 24-48 hours   and then transfer to liquid nitrogen
-80 °C
-190 °C
?. [Culturing]
Freeze back up cells from 6 well plate, place in Mr. Frosty for 24-48 hours  , then transfer to LN2
-190 °C
?. [Clean Up]
Throw away biohazard materials properly
?. [Clean Up]
Clean and sterilize hood with 70% ethanol and turn on UV
?. [Clean Up]
Update cell culture notes in lab notebook
?. [Culturing]
Prepare for KT in T25 flask    a. Take 60-100 ul   of DMSO cell suspension and dilute into 10 mls   of media    b. Spin for 1000 rpm   for 5 mins     c. Aspirate, resuspend in 5 ml   of media, and add to 1xT25 for KT    d. Place flask in incubator (5% CO2, 37oC)     e. Send KT to Cell Line Genetics at 40-50% confluency in about 5-6 days
60 µl
10 mL
Centrifuge: 1000 33
5 mL
37 °C
?. [Observations]
Turn off UV lights and clean hood with 70% ethanol
?. [Observations]
Clean items with 70% ethanol and bring into hood    a. DPBS -/-     b. Sterile Filtered Media     c. Label 2xT150 flasks with ID, date, and p2, add 28 mls   of media per flask    d. 0.05% Trypsin
28 mL