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Passing_Cells

The document outlines a procedure for passing confluent cells from one dish to three dishes to synchronize their growth cycle. It details the materials needed, including PBS, trypsin, and DMEM, as well as step-by-step instructions for rinsing, trypsinizing, and resuspending the cells. Additional notes are provided regarding medium amounts and considerations for transfection.

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Andreea Spiridon
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9 views

Passing_Cells

The document outlines a procedure for passing confluent cells from one dish to three dishes to synchronize their growth cycle. It details the materials needed, including PBS, trypsin, and DMEM, as well as step-by-step instructions for rinsing, trypsinizing, and resuspending the cells. Additional notes are provided regarding medium amounts and considerations for transfection.

Uploaded by

Andreea Spiridon
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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Passing Cells

Description
When cells are confluent, we pass them from one dish to three dishes, to synchronize
the cell growth cycle and prepare for experiment.

Materials
1 PBS

2 Trypsin (in fridge), 1x for smooth muscle cells and 0.5x for endothelial cells
(warm up in water bath)

3 DMEM (with calcium, warm)

4 DMEM with 10% FBS (warm)

5 sterile cell culture dishes (if not tissue culture treated, coat the dishes with 2 %
gelatin (just rinse), if not sterile, incubate with ethanol or light-bath with UV lamp
for 30 min and then rinse with PBS for 3 times. Not necessary for the current
commercial cell culture dish from Fisher).

Procedures
1 Rinse confluent cells with PBS for 3 times

2 Incubate cells with 0.5x trypsin (1 ml for medium dish and 2 ml for large dish),
keep in 37oC for 1.5 min, not to over 2 min.

3 Quickly add DMEM (with calcium) to neutralize trypsin (amount does not really
matter).

4 Pipet some medium to blow cells into suspension. Double check under
microscopy to make sure all the cells are in suspension.

5 Collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the
balance of centrifuger).
6 During the centrifuging period, take 3 new tissue cultured dishes. Label the
dishes with cell name, passage, date, initials of your name.

7 Take out the centrifuged tube containing cells, you should be able to see a
whitish pellet at the bottom of the tube. Tilt the tube and aspirate the supernatant
with vacuum tip, resuspend the cell pellet with 3 ml 10% FBS DMEM by pipetting
up and down 20 times to break cell-cell aggregation. Apply cell solution to
labeled dishes, add more 10% FBS DMEM according to the dish size. (2 ml for
small dish)

8 Swirl the dish gently to allow the cells to spread evenly throughout the dish.

9 Keep the cell dishes in the incubator supplemented with 5% CO2 at 37oC.

Notes
■ The amount of medium can be decided by the size of the cell culture dishes.

■ If you are passing cells for transfection, use DMEM without antibiotics.

■ If passing cells for transfection, make sure there are not too many cells in the plate.
Otherwise, quickly take out some cells before they attach.

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