Sep 11, 2020
Splitting p0 (6wp) to p1 (T75)
- 1University of California, San Francisco
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Andrea Argouarch 2020. Splitting p0 (6wp) to p1 (T75). protocols.io https://dx.doi.org/10.17504/protocols.io.8gjhtun
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 19, 2019
Last Modified: September 11, 2020
Protocol Integer ID: 28907
Abstract
Protocol includes splitting outgrowth of dural cell from a 6 well plate into one T75 flask for expansion.
Materials
STEP MATERIALS
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
GeneMate Cell Scrapers & LifterVWR International (Avantor)Catalog # 490000-254
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Protocol materials
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
GeneMate Cell Scrapers & LifterVWR International (Avantor)Catalog # 490000-254
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
GeneMate Cell Scrapers & LifterVWR International (Avantor)Catalog # 490000-254
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Observations
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.
Cells will grow on the plastic and maybe on the glass coverslip itself
Preparation
Preparation
Turn off UV lights and clean hood with 70% ethanol
Clean items with 70% ethanol and bring into hood
a. Autoclaved # 5 Forceps
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
b. 10 cm dish for forceps, cell scraper, discarded coverslips, and discarded tissue
c. Sterile cell scraper
GeneMate Cell Scrapers & LifterVWR International (Avantor)Catalog # 490000-254
d. DPBS -/-
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
e. Sterile Filtered Media with PenStrep
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
f. Label 1xT75 flask with ID, date, and p1, add 15 mls 15 mL of media per flask
g. 0.05% Trypsin
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
i. Aliquot and warm before use
Culturing
Culturing
Aspirate old media
Rinse by adding 3 mls 3 mL of DPBS per well
Lift coverslips with forceps to allow adequate washing under the coverslip and gently swirl
Aspirate DPBS
Add 2 ml 2 mL of trypsin per well and lift coverslips breifly with forceps
Place in incubator 37 °C for ~5 mins 00:05:00 until cells start to detach
a. Can also gently tap the side of the plate to detach cells from the coverslip
Add 2 mls 2 mL of media per well to inactivate trypsin
With forceps, flip over coverslip within each well
a. Can remove tissue and place in 10 cm dish to discard
Use cell scraper to gently scrap off cells in each well attached to the coverslip
Discard each coverslip in the 10 cm dish
Use cell scraper to scrap off remaining cells (cells that are attached to bottom of plate)
Check under microscope and mark areas with cells still attached with a pen
Re-scrape cell gently if needed
Collect cell suspension in a 50 ml conical
Rinse wells with additional media ~6mls 6 mL to collect any remaining cells
Spin at 1000 rpm 1000 rpm for 5 mins 00:05:00
Re-feed old 6wp plate for backup, 2 mls 2 mL per well
Aspirate supernatant, being careful to not aspirate the pellet
Tap the pellet to resuspend
Add media to a final volume of 5 mls 5 mL and pipette up and down to mix
Add cell suspension to flask
Place flask in incubator (5% CO2, 37oC) 37 °C
Observe and feed the next day to remove debris, then feed every 2-3 days until 90-100% confluent
Clean Up
Clean Up
Throw away biohazard materials properly
a. 10 cm dishes with glass coverslips should be thrown away in biohazard red sharp container
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 and sterilize hood with 70% ethanol and turn on UV
Update cell culture notes in lab notebook