TITLE: Nucleofection of iPSC
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. [Nucleofection - Expand iPSCs]
Split 1 well from a 6 well plate into 3 wells in a 6 well plate.
Cells should be passaged as single cells prior to nucleofection. Split cells into 3 wells of a 6 well plate 48 hours prior to nucleofection (plan for cells to be confluent in 48 hours). You will need 3 million cells per nucleofection, so 3-6 confluent wells should be sufficient
?. [Nucleofection - Expand iPSCs]
Coat 6 well plate with Matrigel (supplemented with RGD fragment) for
1 ml
?. [Nucleofection - Expand iPSCs]
Aspirate media from cell culture.
?. [Nucleofection - Expand iPSCs]
Wash with -of PBS, per well.
1 ml
2 ml
?. [Nucleofection - Expand iPSCs]
Incubate at forto to achieve single cells.
Individual donor cell lines exhibit variable sensitivity to accutase-mediated dissociation. Thus, monitor cells closely to determine when single cell dissociation is achieved.
?. [Nucleofection - Expand iPSCs]
Collect cells in a 5 mL DMEM/F12 and transfer to a 15 mL conical tube.
?. [Nucleofection - Expand iPSCs]
Spin at 750-800 rpm for .
?. [Nucleofection - Expand iPSCs]
Aspirate media.
?. [Nucleofection - Expand iPSCs]
Resuspend cells in mTesR1 (per well) supplemented with 1 uM Rock Inhibitor.
2 ml
?. [Nucleofection - Expand iPSCs]
Plate of cells per well on a Matrigel (supplemented with RGD fragment) coated plate.
2 ml
?. [Nucleofection - Expand iPSCs]
Change media daily with mTesR1
?. [Nucleofection - Expand iPSCs]
Coat plate for nucleofection (2-3 wells) with matrigel supplemented with RGD for
?. [Nucleofection - Expand iPSCs]
Just prior to splitting cells for nucleofection, equilibrate Matrigel/RGD coated plate with 3-5 mL/well DMEM/F12+10% FBS supplemented with 10 uM Rock Inhibitor.
?. [Nucleofection - Expand iPSCs]
Aliquot DNA into 1.7 mL tubes using the table below.  ABCD1  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">pMaxGFP  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">gRNA    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">Cas9 SM168  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">ug/ul    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">3 ug  5  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">100uM    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">3 ul    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">300uM
ABCD1  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#006100">Concentration    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#006100">Volume (uL)    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
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  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">pMaxGFP  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">&nbsp;ul    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">3 ug  5  <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">Donor Oligo    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">100uM    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
  mso-fareast-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:Calibri;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">3 ul    <span lang="EN-US" style="mso-ascii-font-family:Calibri;
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?. [Nucleofection - Split cells for nucleofection]
Aspirate media.
?. [Nucleofection - Split cells for nucleofection]
Wash with - PBS per well.
1 ml
2 ml
?. [Nucleofection - Split cells for nucleofection]
Add of accutase per well.
1 ml
?. [Nucleofection - Split cells for nucleofection]
Incubate at for to minutes (checking at 5 minutes) to achieve single cells.
Individual donor cell lines exhibit variable sensitivity to accutase-mediated dissociation. Thus, monitor cells closely to determine when single cell dissociation is achieved.
?. [Nucleofection - Split cells for nucleofection]
Collect cells in 5 mL PBS and transfer to a 15 mL conical tube.
?. [Nucleofection - Split cells for nucleofection]
Spin at 750-800 rpm for .
?. [Nucleofection - Count cells]
Resuspend cell pellet in PBS in 15 mL conical tube.
3 ml
?. [Nucleofection - Count cells]
Pipette into cell counter.
10 µl
?. [Nucleofection - Count cells]
Using all four corners of the countess slide, calculate the average number of cells.
?. [Nucleofection - Count cells]
Multiply the average by 10,000 (104).
?. [Nucleofection - Count cells]
Multiply product from step 24 by 3 to get the total number of cells._________ average number of cells x 10,000 x 3 = total number of cells.
?. [Nucleofection - Calculate 3 million cells]
Take total number of cells calculated in step 25 and divide by 3 million.
?. [Nucleofection - Calculate 3 million cells]
Take answer from previous step and divide by 3 to get the volume of cells necessary for nucleofection.
?. [Centrifugation]
Transfer the desired volume of cells to microcentrifuge tube.
?. [Centrifugation]
Centrifuge cells at 90 xg for.
?. [Centrifugation]
Aspirate PBS.
?. [Make reaction mix from Lonza Kit]
Make reaction mix from Lonza Kit: P3 Primary Cell 4D (V4XP-3024)- total of per nucleofection.
100 µl
If performing multiple nucleofections, make a master mix.
?. [Make reaction mix from Lonza Kit]
P3 solution
82 µl
?. [Make reaction mix from Lonza Kit]
of Supplement
18 µl
?. [Mixing]
Combine Reaction Mix from step 31 () with previously aliquoted DNA from
100 µl
?. [Mixing]
Mix Reaction Mix and DNA with cell pellet by pipetting up and down with p200 pipette.
Try to pipette as little as possible. Pipette only until mixed.
?. [Mixing]
Transfer to a cuvette.
100 µl
?. [Nucleofection]
Nucleofect with Lonza program CA-137 in P3 solution.
?. [Nucleofection]
Add a small amount of media to cuvette via dropper in order to obtain all the cells form the cuvette.
?. [Nucleofection]
Transfer cells/DNA solution to appropriate pre-coated well containing 2 mL of DMEM/F12+10% FBS+10 uM Rock Inhibitor.
?. [Nucleofection]
Incubate at overnight.
?. [Post Nucleofection]
Continue cultruing the iPSC in 1 well of a 6 well plate for 5-7 days post nucleofection, changing mTesR1 daily.     a.  post nucleofection--add mTesR1 with 5 uM Rock Inhibitor.    b. post nucleofection-- add mTesR1 with 2.5 uM Rock Inhibitor.    c.  post nucleofection-- add mTesR1 with 1uM Rock Inhibitor.
24 hours after nucleofection, cells exhibit extended processes - this is expected. Over the following 24-72 hours, cells recover and return to typical rounded iPSC morphology. The exact timeline for the morphological recovery is dependent on donor lines.
?. [Post Nucleofection]
Continue cultruing in mTesR1 until confluent.
Cells must be maintained for 5 days post-nucleofection prior to screening to minimize chimeric clones.