Apr 25, 2018
Basic Cell Culture Maintenance: Plating Cells
- 1Oregon Health and Science University
- Ellison Lab
Protocol Citation: Britt DK Gratreak 2018. Basic Cell Culture Maintenance: Plating Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.pqgdmtw
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: April 25, 2018
Last Modified: April 25, 2018
Protocol Integer ID: 11752
Keywords: HEK293, cell culture, cells, plate, plating, 12 well, 12 well plate, seed, seeding, ellison lab
Abstract
Basic protocol to plate Human Embryonic Kidney 293 (HEK293) cells in to 12 well plates. This protocol can be modified easily to plate in different volumes and concentrations.
Materials
MATERIALS
HyClone Classical Liquid Media Dulbeccos Modified Eagles Medium (DMEM)Fisher ScientificCatalog #SH3024301
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
Falcon™ Polystyrene Microplate (12 well)Fisher ScientificCatalog #08-772-29
STEP MATERIALS
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
Protocol materials
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
HyClone Classical Liquid Media Dulbeccos Modified Eagles Medium (DMEM)Fisher ScientificCatalog #SH3024301
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
Falcon™ Polystyrene Microplate (12 well)Fisher ScientificCatalog #08-772-29
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
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 |
Split Cells
Split Cells
Split cells following the Basic Cell Culture Maintenance: Splitting Cells protocol. Cells leftover from split flasks will be used to count and plate cells.
Counting Cells
Counting Cells
Add 75 μL media and 25 μL leftover cells into a 1.5 mL microcentrifuge tube. (1:4 dilution)
Note
To avoid contamination, it is best practice to aliquot a small amount of the media for this purpose by using a sterile serological pipette. Always avoid using a micropipette to directly make contact with the media stock.
Mix thoroughly by flicking.
Load 10 μL into the hemacytometer and view under microscope.
Hausser Scientific Bright-Line™ Counting ChamberFisher ScientificCatalog #02-671-51B
Calculating Concentration
Calculating Concentration
Count cells per quadrant. Average them. Multiply by 10,000 and the dilution factor (4).
Note
For example, if you counted 66, 65, 58, and 70 cells in their individual quadrants, they would average to 64.75 cells per quadrant. Multiply this by 10,000 and then by 4. This will lead to determining a concentration of 2,590,000 cells per mL in the leftover cells from splitting.
Creating a Master Mix
Creating a Master Mix
Calculate amount of cells + new media needed to create a master mix using C1V1, in which concentration is cells per mL/cells per well. (one well = 1 mL) Add 3 more wells of volume than needed to calculations to account for errors.
Note
Using the example previously, 2,590,000 cells per mL is the concentration of cells that are leftover from splitting. The amount of cells to plate depends on the purpose for the cells and the rate of growth of the cell line. Typically, Lipofectamine 2000 transfection with HEK293 cells works best with plating 500,000 cells per well. For a 12 well plate, add 3 extra wells to account for any pipetting error/interfering bubbles.
The equation to solve for would be:
'HAVE' vs 'WANT'
C1V1 = C2V2
2,590,000 cells/mL * (X mL) = 500,000 cells/mL * (15 mL)
X mL = (500,000 cells/mL * (15 mL)) / 2,590,000 cells/mL
X = 2.9 mL cells
15 - 2.9 mL = 12.1 mL media
Thus, your master mix would consist of:
2.9 mL cells
+ 12.1 mL media
= 15 mL total.
Create master mix in 50 mL falcon tube. Invert master mix several times just before plating to ensure it is evenly mixed.
Plating
Plating
Label plates with strain, date, and initials.
Add 1 mL master mix to each well.
Incubate
Incubate
Gently mix plates with gentle shaking. Return the cells to the incubator at 37°C and 5% CO2.