Sep 10, 2023
Basic Cell Culture Maintenance: Splitting Cells
Forked from Basic Cell Culture Maintenance: Splitting Cells
This protocol is a draft, published without a DOI.
- 1Technion
Protocol Citation: leroi.bwh 2023. Basic Cell Culture Maintenance: Splitting Cells. protocols.io https://protocols.io/view/basic-cell-culture-maintenance-splitting-cells-czsmx6c6
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: September 09, 2023
Last Modified: September 10, 2023
Protocol Integer ID: 87597
Keywords: cell culture, hek293, cell, maintenance, split, cell splitting, splitting, maintaining cell lines, cell lines, maintain, cells
Abstract
Basic protocol to split THP1
Materials
MATERIALS
HyClone Classical Liquid Media Dulbeccos Modified Eagles Medium (DMEM)Fisher ScientificCatalog #SH3024301
Gibco™ (Phosphate Buffered Saline) Solution, pH 7.4 (PBS)Fisher ScientificCatalog # 10010-049
Trypsin-EDTA (0.25%), phenol redThermofisherCatalog #25200-056
Safety warnings
- Human Embryonic Kidney (HEK293) cells are biosafety level 2 (BSL-2) and should be handled according to the CDC's Biosafety in Microbiological and Biomedical Laboratories (BMBL) guidelines. They are considered BSL-2 not because they are inherently hazardous or infectious, but because of their potential to be infected with pathogens and in turn infect their handlers. Due to the impossibility to regularly screen this cell like for every human pathogen, HEK293 cells should always be handled as potentially infectious. Other BSL-2 cell lines include those positive for Legionella pneumophila, HIV, and other disease-causing pathogens in humans.
- Dispose of ALL waste that comes into contact with cells such as pipettes, gloves, and materials as biohazardous waste.
- Bleach all direct cell waste thoroughly. In our lab, our vacuum line tube empties in to a sealed waste jug with bleach already added to the bottom of it, making up at least 10% of the total volume. This way, aspirated media and cells immediately come into contact with the bleach. Before disposing of glass pipettes, we aspirate a small amount of 10% bleach through to clean both the pipette and tubing, then dispose of the pipettes as biohazardous sharps.
Before start
Make complete DMEM:
| Reagent | Volume | |
| DMEM | 432.5 mL | |
| FBS | 50 mL | |
| Pen/Strep | 5 mL | |
| HEPES (1M, pH 7.4) | 12.5 mL |
Preparation
Preparation
Confirm that cells are at least 80% confluent by microscopy.
Warm complete RPMI in 37°C water bath.
UV light for 30 minutes then spray down the biosafety cabinet with 70% ethanol and use as a secondary decontaminant.
Prepare 20 µL of Trypan blue into a 200 µl Eppendorf.
Remove Media
Remove Media
Aspirate the media from the flask using a sterile autoclaved glass pipette. Do not touch the cells with the pipette.
Note
To avoid touching cells, is best to tilt the flask and gently remove media from a corner.
Transfer
Transfer
Transfer ALL contents/cells to a 50 mL falcon tube.
Spin
Spin
Spin down 130 rcf for 7 minutes.00:07:00 130 x g, 37°C
7m
While spinning, clean surfaces with EtOH and label new flasks, noting the +1 passage number and dilution.
Remove Media
Remove Media
Aspirate media from falcon tubes with cells; make sure to not disturb the pellet.
Resuspend Pellet
Resuspend Pellet
Add 5 mL media to pellet and pipette violently up and down.
Count the cells
Count the cells
Pipette 20 µL and add them to the Trypan blue
Count the cells using a Neubauer counting chamber
Calculate the required amount of medium to keep a concentration of 100 kC/ml
Prepare New Flask
Prepare New Flask
Add the required amount of medium.
Distribute in flasks, no more than 50 mL per flask.
Incubate
Incubate
Gently shuffle, ensure even dispersal, and return the fresh flask to incubator.