Sep 11, 2020
Splitting p1 (1xT75) to p2 (2xT150)
- 1University of California, San Francisco
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Andrea Argouarch 2020. Splitting p1 (1xT75) to p2 (2xT150). protocols.io https://dx.doi.org/10.17504/protocols.io.8gnhtve
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: 28910
Abstract
Protocol includes splitting growing cell line from a T75 flask into two T150 flasks for expansion.
Materials
STEP MATERIALS
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
Protocol materials
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
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
Observations
Observations
At 90-100% confluency, split T75 flask into 2xT150 flasks
Preparation
Preparation
Turn off UV lights and clean hood with 70% ethanol
Clean items with 70% ethanol and bring into hood
a. DPBS -/-
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
b. Sterile Filtered Media
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
c. Label 2xT150 flasks with ID, date, and p2, add 28 mls 28 mL of media per flask
d. 0.05% Trypsin
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Culturing
Culturing
Aspirate old media
Rinse by adding 5 mls 5 mL of DPBS per flask and gently swirling
Aspirate DPBS
Add 2 mls 2 mL of trypsin per flask and place in incubator 37 °C for 2-3 mins 00:03:00 until cells are stating to detach
a. Can also gently tap the side of the flask to detach cells
Check under microscope
Add 10 mls 10 mL of fibroblast media per flask to inactivate trypsin
Collect cell suspension in a 15 ml conical
Spin at 600 rpm 600 rpm for 4 mins 00:04:00
Aspirate supernatant, being careful to not aspirate the pellet
Tap the pellet to resuspend
Add fibroblast media to 4 mls 4 mL
Add 2 ml 2 mL of cell suspension per flask
Place flask in incubator (5% CO2 , 37oC 37 °C )
Observe and feed the next day, then feed every 2-3 days until 90-100% confluent
Clean Up
Clean Up
Throw away biohazard materials properly
Clean and sterilize hood with 70% ethanol and turn on UV
Update cell culture notes in lab notebook