Nov 23, 2023

Passaging cells in MultiFlasks

This protocol is a draft, published without a DOI.
  • 1Cancer Research UK Cambridge Institute, University of Cambridge
Allan JW Lui
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Protocol Citation: Allan JW Lui 2023. Passaging cells in MultiFlasks. protocols.io https://dx.doi.org/
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: December 06, 2022
Last Modified: November 23, 2023
Protocol  Integer ID: 73594
Keywords: multiflask, cells in multiflask, passaging cell
Abstract
Simple protocol for working with Falcon Multi-Flasks
Materials
Falcon 3-layer or 5-layer Multi-Flasks (Corning 353143 or Corning 353144, respectively)
5-layer Multi-Flask
3m
Per flask, Prepare and pre-warm:
  • 200ml PBS
  • 30ml 0.25% Trypsin-EDTA or other detachment reagent
  • 70ml complete media for trypsin inactivation, plus extra for reseeding
Pour out media in flask, wash out remaining media twice by:
  1. Adding 100 mL PBS
  2. Distributing equally and evenly between layers to cover the entire culture surface area
  3. Pouring out PBS


Add 30 mL 0.25% Trypsin and distribute equally between layers
Incubate 00:03:00 37 °C

3m
Inactivate trypsin with 30 mL media distributed equally between layers
Collect cell suspension into 50ml tubes or 100ml bottles
Wash out remaining cells with 40 mL media

Centrifuge at 200 x g, 00:05:00 , remove trypsin, resuspend in fresh media if necessary, then count cells and reseed

5m
Per flask, Prepare and pre-warm:
Reagent2-layer5-layer
PBS250ml500ml
Trypsin40ml100ml
Complete media for inactivation80ml200ml
Complete media for plating220ml550ml