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Cell Passaging

The document provides instructions for cell passaging, seeding, dosing, and adding cell substrate. Key steps include coating plates, removing old media, washing cells, treating with trypsin, centrifuging, resuspending cells, counting cells, seeding cells, adding standards and samples to wells, and adding substrate.

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Michelle Liau
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29 views2 pages

Cell Passaging

The document provides instructions for cell passaging, seeding, dosing, and adding cell substrate. Key steps include coating plates, removing old media, washing cells, treating with trypsin, centrifuging, resuspending cells, counting cells, seeding cells, adding standards and samples to wells, and adding substrate.

Uploaded by

Michelle Liau
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Cell Passaging

1. GR and ? need to coated with gel trex


2. Discard all the old media
3. Wash with 5ml DPBS and discard
4. Add 0.05% trypsin for all the cells, except 0.25% trypsin for HEPG2
5. Put at incubator for 3 min.
6. Add 8ml of media if check the cells have successfully detached
7. Move all the suspension into the tube and centrifuge for 5 min (0.02rpm for all cells, except
0.01rpm for HEPG2)
8. In the meanwhile, add 12ml of media to the new flask and write down the cell’s name and
presage number and date.
9. After centrifugation, take out all the suspension (but be careful of the white pellets) and hit
the tube hard.
10. Calculate for the passaging ration and resuspended to the regarded amount.
11. Add the calculated amount the new flask (with media) and check under the microscope.

Seeding

1. After resuspended, take out 100ul of the mixed solution and mixed with 100ul of dye in small
tube.
2. In normal situation, we use the cell counter machine to count. It will tell the amount of cell to
mix with the amount of assay media.
3. The manual count for cells is ? × 10! /ml and count for the amount of cells need. Use 384
wells (each well is 20ul (?)) and amount of assay media = total amount – amount of cells
needed.
4. After seeding, settle in the incubator for 45 minutes and check under the microscope. And put
at the incubator for 6 hours for all the cells, except 5 hours for ARE.

Dosing

• Always 10 points for standards.


• 4 wells for controls, 2 wells for assay media, 1 well for blank (with 2.5% DMSO).
1. Count for wells that needed for 2.5% DMSO (except the first well for standard and samples)
[40x dilution] and remember vortex.
2. If 40x dilution, then the first wells for samples (4ul) + assay media (156ul), hence is 2.5%
DMSO.
Standards
Assay 80 ul 80 ul 80 ul 80 ul Assay 80 ul 80 ul 80 ul 80 ul
media 2.5% 2.5% 2.5% 2.5% media 2.5% 2.5% 2.5% 2.5%
156ul + DMSO DMSO DMSO DMSO 156ul + DMSO DMSO DMSO DMSO
sample 1 sample 1
4ul 4ul
Assay 80 ul 80 ul 80 ul 80 ul Assay 80 ul 80 ul 80 ul 80 ul
media 2.5% 2.5% 2.5% 2.5% media 2.5% 2.5% 2.5% 2.5%
156ul + DMSO DMSO DMSO DMSO 156ul + DMSO DMSO DMSO DMSO
sample 2 sample 2
4ul 4ul
Assay 80 ul 80 ul 80 ul 80 ul Assay 80 ul 80 ul 80 ul 80 ul
media 2.5% 2.5% 2.5% 2.5% media 2.5% 2.5% 2.5% 2.5%
156ul + DMSO DMSO DMSO DMSO 156ul + DMSO DMSO DMSO DMSO
sample 3 sample 3
4ul 4ul
3. After adding the DMSO and samples in 96 wells, then take 80ul from first well to 2nd well
and accordingly, and hence the last well always has the largest amount.
4. Add 5ul of chemical to the 384 wells and put at incubator for 24hrs.

Calculation:

Cell (in the 96 wells) Cell + Chemical Substrate


(How many times dilution (Fixed 5x dilution) (Fixed 6x dilution)
depends on the total dilution)

• For example: Total dilution= 200x dilution


∴ Hence we do 40x dilution at the 96 wells.

• And for the 5x dilution, 24ul cell + 6ul chemical = 30 ul (total amount)
• And for the 6x dilution, 30 + 6ul substrate = 36ul

Adding cell substrate


• 6ul of A + 60ul B + 934ul C = 1ml (base amount)
• Remember to turn off the light (the substrate is light sensitive)
• After adding the substrate, must keep in the box for 2hrs ( but depends on which cell lines) in
room temperature.

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