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Passaging Protocol

The Passaging Protocol outlines the steps and supplies needed for subculturing cells based on Lonza documentation. It includes detailed instructions for preparing reagents, detaching cells, and transferring them to new flasks, with emphasis on maintaining proper temperatures and conditions. The protocol also provides guidance on cell counting and seeding density for optimal growth.

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7 views2 pages

Passaging Protocol

The Passaging Protocol outlines the steps and supplies needed for subculturing cells based on Lonza documentation. It includes detailed instructions for preparing reagents, detaching cells, and transferring them to new flasks, with emphasis on maintaining proper temperatures and conditions. The protocol also provides guidance on cell counting and seeding density for optimal growth.

Uploaded by

spoorvi464
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Passaging Protocol:

Based on Lonza documentation

Supplies:
• Trypsin-EDTA (2mL for each 25 cm2), room temperature
• Trypsin neutralizing solution (4 mL for each 25 cm2), room temperature
• Lonza HEPES-BSS (7 mL for each 25 cm2), room temperature. Can use PBS if out of
HEPES-BSS.
• Growth medium (5 mL per 25 cm2), room temperature
• New flasks
• Centrifuge tube (15 or 50 mL depending on number of flasks)
• To count cells (if desired, see separate protocol for instructions): hemocytometer,
trypan blue, glass slides, small Eppendorf tube

Steps:
1. Passage after reaching 70-80% confluence
2. For each 25 cm^2 of cells to be subcultured:
a. Thaw 2 mL of trypsin/ETDA to allow to come to room temperature
b. Allow 7-10 mL HEPES buffered saline solution to come to room temperature
c. Allow 4 mL Trypsin neutralizing solution to come to room temperature
d. Remove growth medium from fridge and start warming to room temperature.
3. For each flask, aspirate spent media without disturbing monolayer
4. Rinse cell layer with 5 mL HEPES-BSS to remove residual traces of serum. Then remove
and discard HEPES-BSS.
5. Add room temperature Trypsin-EDTA solution (2 mL for every 25 cm^2) to each flask
6. Gently rock the flask to ensure complete coverage over the cells.
7. Observe under the microscope. When cells pull away from each other and round up
(usually 2-5 min), remove flask from under the microscope and gently tap it from several
side to promote detachment of the cells from the flask surface.
a. If only a few cells detach, you may not have let them trypsinize long enough.
Wait 30 seconds and rap again. If cells still do not detach, wait and rap every 30
seconds thereafter.
b. If the majority of cells do not detach within seven minutes, the trypsin is either
not warm enough or not active enough to release the cells. Either re-trypsinize
with fresh, warm Trypsin/EDTA solution or rinse with Trypsin Neutralizing
Solution and then add fresh, warm medium to the culture vessel and return to
an incubator until fresh trypsinization reagents are available.
8. When majority of cells are detached, quickly add trypsin neutralizing solution to each
flask. Gently swirl.
9. Transfer dissociated cells to centrifuge tube (15 mL or 50 mL tube depending on number
of flasks you’re passaging). Rinse the flask with a final 2 ml of HEPES-BSS to collect
residual cells, and add this rinse to the centrifuge tube.
10. Examine the harvested flask under the microscope to make sure the harvest was
successful by looking at the number of cells left behind. This should be less than 5%.
(Can skip if confident that most cells lifted off based on visualization in step 8).
11. Centrifuge cells at 220 xg for 5 min.
a. Aspirate most of the supernatant, except for 100-200 μl to avoid disturbing
pellet.
12. Re-suspend in 1 mL medium, and count cells (if desired). See “Cell Counting using
Hemocytometer” protocol for counting instructions.
a. Determine the total number of flasks to inoculate by using the following
equation. The number of flasks needed depends upon cell yield and seeding
density (Recommended seeding density for fibroblasts is 2500-5000 cells/cm2).

13. Prepare subculture flasks (5 mL media for every 25 cm^2)


14. Seed new flasks. Use the following equation to calculate the volume of cell suspension
to seed into your flasks:

15. Place freshly seeded flasks in 37 degC, 5% CO2 incubator for 24-48 hours before
processing cells further.

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