Haynes Lab:Notebook/Jan/2015/03/30: Difference between revisions
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==Tissue Culture Training== | ==Tissue Culture Training== | ||
* Passaging - U2OS, split PS3 --> PS4 | * Passaging - U2OS, split PS3 --> PS4 | ||
6-well plate - U2OS | *6-well plate - U2OS | ||
Passaging - U2OS | *Passaging - U2OS | ||
Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells] | *Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells] | ||
Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S | *Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S | ||
Use 90-100% confluent T-75 | *Use 90-100% confluent T-75 | ||
Make 1 new T-75 at a 1:10 dilution | *Make 1 new T-75 at a 1:10 dilution | ||
Passage number: 4 | *Passage number: 4 | ||
6-well plate - U2OS | * 6-well plate - U2OS | ||
Use same resuspension from old flask in Passaging - U2OS | * Use same resuspension from old flask in Passaging - U2OS | ||
Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration | *Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration | ||
Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium | *Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium | ||
Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well | *Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well | ||
Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well | *Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well | ||
Make a batch of diluted cells for new 6-well plate: | *Make a batch of diluted cells for new 6-well plate: | ||
Into a sterile 50 mL conical add... | *Into a sterile 50 mL conical add... | ||
(6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells | *(6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells | ||
(6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium | *(6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium | ||
* Passaging - KAH126, split PS3 --> PS4 | * Passaging - KAH126, split PS3 --> PS4 | ||
6-well plate - KAH126 | *6-well plate - KAH126 | ||
Passaging - KAH126 | *Passaging - KAH126 | ||
Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells] | *Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells] | ||
Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S | *Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S | ||
Use 90-100% confluent T-75 | *Use 90-100% confluent T-75 | ||
Make 1 new T-75 at a 1:10 dilution | *Make 1 new T-75 at a 1:10 dilution | ||
Passage number: 4 | *Passage number: 4 | ||
Dox induction - KAH126 | *Dox induction - KAH126 | ||
Induce cells in 6-well plate with dox to express PcTF (RFP) | *Induce cells in 6-well plate with dox to express PcTF (RFP) | ||
Stock concentration of dox = 1.0 mg/mL | *Stock concentration of dox = 1.0 mg/mL | ||
Desired final concentration of dox = 1.0 μg/mL (1000x diluted) | *Desired final concentration of dox = 1.0 μg/mL (1000x diluted) | ||
Therefore add 1.0 μL of stock dox for every 1.0 mL medium in well | *Therefore add 1.0 μL of stock dox for every 1.0 mL medium in well | ||
4.0 mL medium in well x 1.0 μL stock dox = 4.0 μL stock dox to be added to each well | *4.0 mL medium in well x 1.0 μL stock dox = 4.0 μL stock dox to be added to each well | ||
Revision as of 10:12, 30 March 2015
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Tissue Culture Training
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