Apr 29, 2026

RPE1 subculture

This  protocol  is a draft, published without a DOI.
  • 1Discovery Research Platform for Hidden Cell Biology
Victoria Munro
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Protocol Citation: Georg Kustatscher 2026. RPE1 subculture. 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: In development
We are still developing and optimizing this protocol
Created: June 21, 2024
Last Modified: April 29, 2026
Protocol  Integer ID: 102222
Keywords: rpe1 cell, warm reagent, cell, subculture
Abstract
Guidance for subculture of RPE1 cells

Notes: warm reagents to 37oC before culturing 
Materials
DPBS, no calcium, no magnesium (Gibco, 14190094)
Trypsin-EDTA (0.5%), no phenol red (Gibco, 15400054)
-> dilute to 0.05% in DPBS without calcium and magnesium for use
Trypan blue (BIO-RAD, 1450021)
Cell Counting Slides for TC10‱/TC20‱ Cell Counter (BIO-RAD, 1450015)
CELL CULTURE FLASK, 50 ML, 25 CM² (Greiner, 690175)
CELL CULTURE FLASK, 250 ML, 75 CM² (Greiner, 658175)
CELL CULTURE FLASK, 550 ML, 175 CM² (Greiner, 660160)


Grow cells to appropriate confluency (e.g 70%)
Remove media and wash 2x with DPBS without Ca2+ / Mg2+
Add trypsin-EDTA (0.05%; 300ul for T25, 1ml for T75, 2-3ml T175), rotate flask to ensure cell monolayer is covered
Incubate for approx. 5 min in cell incubator
Examine cells to ensure they are all detached, can gently tap side of flask to aid detachment
Resuspend in media (at least x2 trypsin vol for quenching) and either perform a cell count using trypan blue exclusion to determine cell number for seeding or take a pre-determined portion
  • e.g between 1/10 and 1/15 for 3 day split or between 1/20 and 1/25 for 4 day split into flask of the same size
Transfer cells to a new flask with pre-warmed medium
  • T25: 5ml
  • T75: 12ml
  • T175: 35ml
Repeat as appropriate
  • e.g split every Monday and Thursday for 3 and 4 day split respectively