https://wiki.tanakalab.org/index.php?title=Special:NewPages&feed=atom&hideredirs=1&limit=50&offset=&namespace=0&username=&tagfilter=&size-mode=max&size=0Tanaka Wiki - New pages [en]2024-03-29T11:33:12ZFrom Tanaka WikiMediaWiki 1.35.8https://wiki.tanakalab.org/index.php/Frog_Prrx1:CreER_line_screeningFrog Prrx1:CreER line screening2022-07-18T23:52:15Z<p>Tzi.lin: </p>
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<div>Transgenic TLM115 Frog Screening Protocol 2022.07.18 Tzi-Yang<br />
<br />
A. Mating (ask Birgit if not clear), ~1 hour<br />
*Choose the mating pairs and make notes on the frog list. <br />
**E.g. TLM115_F_02 x C36_107M_201M <br />
*Thaw hCG stored in the -20 degree freezer (Xenopus drawer).<br />
*Bring 20L tank(s) and lids in the frog exp room with a trolley (1 tank per mating pair).<br />
*Prepare 7-8L 20mM NaCl per tank:<br />
**5M NaCl 26uL per tank<br />
**Add tap water to 7-8L mark on the tank (or roughly 2/5 to 1/2 of the tank) <br />
*Find animals in the colony using the chip reader or photos.<br />
*Put the pairs together in a tank and label the tank.<br />
*Inject hormones and put the pairs back to the tank: <br />
**400-500unit hCG per female<br />
**40-50unit hCG per male<br />
*Cover the tank with a carton box or an orange bag.<br />
(Leave the tank on the trolley at the corner of the frog exp room overnight.<br />
<br />
B. Collecting eggs (The next day afternoon), ~1-2hr <br />
*Print labels.<br />
*Collect the eggs to a white box with tap water.<br />
*If the egg clumps together, separate them into smaller clumps.<br />
**Dejellying and picking the surviving embryos is recommended.<br />
**Collect ~500 embryos in 2 boxes would be enough.<br />
*Change water at least every other day!<br />
<br />
C. First Screening at 7-8 days after mating. ~1-2 hours<br />
**The timing is important because 1) the embryos should be floating but not active swimming at these stages, so we do not need to anesthetize them. 2) and it is the earliest timing when the trunk-GFP and lens-Cherry signals could be easily distinguished. <br />
*First screen for trunk-GFP+ embryos using a plastic dropper under the fluorescence stereoscope.<br />
*Then screen for lens-mCherry+ embryos in the trunk-GFP+ embryos. <br />
*Grow the double positive animals and label +/+ on the white box.<br />
**Start feeding only from 14 days or later. Feeding too early and feeding too much will kill them.<br />
<br />
D. 4OHT treatment: ~15-25 days after mating, ~30min/per treatment<br />
*Thaw a tube of 4OHT stock (20mM in DMSO) in dark.<br />
*Prepare 1.5uM 4OHT in 0.1xMMR in a genotyping box (200mL per ~100 animals)<br />
**4OHT (20mM) 15uL<br />
**10xMMR 2mL<br />
**MilliQ water to 200mL<br />
*Transfer the embryos into the 4OHT solution. Keep the box in dark overnight.<br />
*(The next day, ~18-22 hours later) Wash and transfer the animals to tap water.<br />
**Feed and clean daily until the next treatment.<br />
**To get the best conversion result, do 2x treatments per week for two weeks. <br />
**Do not put them into the system during the treatment period. Wait at least 5-7 days from the last treatment.<br />
<br />
E. Second screening: >7 days after the last treatment, or until stage >52 (~2 month old).<br />
*Anesthetize the animals briefly <br />
*Screen for Cherry+ limbs under the fluorescence stereoscope.<br />
<br />
*Grow the converted animals. <br />
*Label the tank and the frog list properly about the result of 4OHT treatment.<br />
**The best screening stage is between stage 51-53.<br />
**The Cherry signal could be observed in the limb mesenchyme and the mid brain regions. Occasionally the Cherry signal could be found in tail muscle.<br />
**IMPORTANT: there will be ~ 30% of the double positive animals that do not have conversion in the limb. The reason is yet to be determined. So always screen properly!<br />
*Grow the converted animals up and use them for experiments.<br />
<br />
F. (Optional) Third screening: stage 66 (~ 6-7months old)<br />
*Keep the animals with strong conversion and clean conversion result for the next generation. Strong conversion means the Cherry signals are strong in the joints. Clean conversion means no muscle were labeled in the froglet leg. Below is an example.</div>Tzi.linhttps://wiki.tanakalab.org/index.php/For_cell_transplantationFor cell transplantation2022-07-18T23:09:56Z<p>Tzi.lin: /* Cell transplantation */</p>
<hr />
<div>= Cell transplantation =<br />
----<br />
<li>(Continuing directly from [[LiberaseTM cell dissociation protocol|Liberase dissociation]] or from [[for FACS|FACS]])</li><br />
*Resuspend the cell pellet with 100-200uL AMEM(0) depending on the size of the pellet.<br />
*Count the cells using a hemocytometer.<br />
**Standard protocol: 5uL cell solution + 5uL trypan blue, count the cells in 5 blocks<br />
::: cell number/5 x 20 = cell number/uL<br />
*Calculate the required volume (<200uL) for transplantation: 10k cells per transplant.<br />
**For example, minimum 50k donor cells are required for 5 transplants.<br />
**If the required volume is larger than 200uL, spin down and resuspend the cells with a smaller volume.<br />
*Load the required volume of the donor cell solution into a sealed yellow tip.<br />
*Centrifuge the sealed yellow tips in a 50mL falcon tube. 300xrcf, 20 degree, 5 minutes.<br />
*Follow the following protocol from here.<br />
<br />
[[File:text1-1.png|400px|thumb|left| ]]<br />
[[File:text1-2.png|400px|thumb|left| ]]<br />
[[File:text2.png|400px|thumb|left| ]]<br />
[[File:text3.png|400px|thumb|left| ]]<br />
[[File:Screenshot 2022-07-19 at 01.18.01.png|200px|thumb|left| ]]<br />
[[File:2.png|200px|thumb|left| ]]<br />
[[File:3.png|400px|thumb|left| ]]</div>Tzi.linhttps://wiki.tanakalab.org/index.php/LiberaseTM_cell_dissociation_protocolLiberaseTM cell dissociation protocol2022-07-18T22:33:17Z<p>Tzi.lin: /* Liberase TM cell dissociation on amphibian limb tissue */</p>
<hr />
<div>= Prepare Reagents =<br />
----<br />
=== 0.7x PBS ===<br />
*Dilute 1x PBS to 0.7x with dH2O<br />
:-> Chill on ice before use.<br />
<br />
=== Liberase TM solution ===<br />
*40uL Stock Liberase TM (100x, 26WU/mL)<br />
*3960uL 0.7xPBS in a FACS tube<br />
:-> Chill on ice before use.<br />
<br />
=== AMEM(0) (serum-free medium) ===<br />
*114.3mL F12:DMEM+Glutmax<br />
*1.5mL Sodium pyruvate (100mM)<br />
*1.5mL B27 supplement<br />
*1.5mL Insulin (1 mg/mL)<br />
*1.5mL MEM Non-Essential Amino Acid (NEAA)<br />
*1.5mL Penicillin/Streptomycin (10000 U/mL)<br />
*28.2mL Sterile ddH2O<br />
:-> Total 150mL, filter. <br />
:-> Chill on ice before use.<br />
<br />
=== High-Serum AMEM (HS-AMEM) ===<br />
*125mL Minimum essential medium (MEM)<br />
*20mL Heat-inactivated fetal bovine serum (56 degree, 30 min)<br />
*2mL Insulin (1 mg/mL)<br />
*2mL Glutamine (200mM)<br />
*2mL Penicillin/Streptomycin (10000 U/mL)<br />
*49mL Sterile ddH2O<br />
:-> Total 200mL, filter. <br />
:-> Chill on ice before use.<br />
<br />
= Liberase TM cell dissociation on amphibian limb tissue =<br />
----<br />
*Collect the limb tissue to dissociate into a new 100mm dish.<br />
*Wash the tissue in a 60mm dish with 0.7x PBS to remove blood and debris.<br />
*Transfer the tissue into a new 60mm dish with fresh 0.7x PBS.<br />
*Remove the skin.<br />
*Transfer the limb mesenchyme into a new 60mm dish. Remove as much liquid as possible.<br />
*Chop the tissue into <500mm^3 cubes with a #21 scalpel.<br />
*Transfer the tissue pieces into the Liberase TM solution.<br />
*Rotate on a wheel 40-50 minutes, room temperature. Vigorously shake every 15-20 minutes to disperse the tissue.<br />
**The tissue will start to clump together after ~20 minutes.<br />
*Put the tube on a rack and wait until the remaining tissue pieces to settle down.<br />
*Collect the cleared, top 3mL solution and filter through a 30mm filter cup on a 15mL falcon tube.<br />
*Mechanically dissociate the remaining tissue by pipetting the remaining 1mL solution 15-20 times with a P1000 tip.<br />
*Filter the remaining solution with the same 30mm filter cup.<br />
*Stop the enzymatic reaction by washing the FACS tube with 4mL HS-AMEM, and then filter the HS-AMEM with the same 30mm filter cup.<br />
*Collect the remaining solution at the bottom of the filter cup into the 15mL falcon tube, gently mix.<br />
::-> If doing scRNA-seq directly, immediately filter through 10um filter cup. Then continue.<br />
*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.<br />
*Wash the cell pellet with 5mL 0.7xPBS<br />
*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.<br />
::-> If doing FACS, continue with [[for FACS|FACS protocol]].<br />
*Wash the cell pellet with 5mL 0.7xPBS<br />
*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.<br />
::-> If doing scRNA-seq directly, continue with the [[for scRNA-seq|scRNA-seq protocol]].<br />
::-> If doing cell transplantation directly, continue with the [[for cell transplantation|transplantation protocol]]</div>Tzi.linhttps://wiki.tanakalab.org/index.php/For_scRNA-seqFor scRNA-seq2022-07-18T22:23:21Z<p>Tzi.lin: /* scRNA-seq */</p>
<hr />
<div>= scRNA-seq =<br />
----<br />
<li>(Continuing directly from [[LiberaseTM cell dissociation protocol|Liberase dissociation]] or from [[for FACS|FACS]])</li><br />
*Resuspend the cell pellet with 50-100uL AMEM(0) depending on the size of the pellet.<br />
*Count the cells using a hemocytometer.<br />
**Standard protocol: 5uL cell solution + 5uL trypan blue, count the cells in 5 blocks<br />
::: cell number/5 x 20 = cell number/uL<br />
**Ideal concentration is between 800-1200 cells/uL <br />
**If there are too many doublets or clumps, better repeat the experiment.<br />
**If the concentration is too low or too high -> spin down the cells and resuspend in a different volume<br />
*Follow the 10X scRNA-seq protocol from here, or submit the samples to the NGS facility.</div>Tzi.linhttps://wiki.tanakalab.org/index.php/For_FACSFor FACS2022-07-18T22:14:40Z<p>Tzi.lin: /* FACS */</p>
<hr />
<div>= FACS =<br />
----<br />
<li>(Continue from the [[LiberaseTM cell dissociation protocol]])</li><br />
*Resuspend the cell pellet with 150-300uL AMEM(0) depending on the size of the pellet.<br />
*Immediately sort the cells into 1.5mL tube with 700uL AMEM(0).<br />
**Normally we use F02 sorter because it has lasers for both GFP and mCherry.<br />
*Note the FACS count.<br />
*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.<br />
<br />
*The sample is ready for downstream applications:<br />
::-> [[For scRNA-seq|scRNA-seq]] <br />
::-> [[For cell transplantation|Cell transplantation]]</div>Tzi.linhttps://wiki.tanakalab.org/index.php/LiberaseTM_cell_dissociation_for_axolotl/frog_limb_cellsLiberaseTM cell dissociation for axolotl/frog limb cells2022-07-18T21:40:16Z<p>Tzi.lin: /* Liberase TM cell dissociation on amphibian limb tissue */</p>
<hr />
<div>= Prepare Reagents =<br />
----<br />
=== 0.7x PBS ===<br />
*Dilute 1x PBS to 0.7x with dH2O<br />
:-> Chill on ice before use.<br />
<br />
=== Liberase TM solution ===<br />
*40uL Stock Liberase TM (100x, 26WU/mL)<br />
*3960uL 0.7xPBS in a FACS tube<br />
:-> Chill on ice before use.<br />
<br />
=== AMEM(0) (serum-free medium) ===<br />
*114.3mL F12:DMEM+Glutmax<br />
*1.5mL Sodium pyruvate (100mM)<br />
*1.5mL B27 supplement<br />
*1.5mL Insulin (1 mg/mL)<br />
*1.5mL MEM Non-Essential Amino Acid (NEAA)<br />
*1.5mL Penicillin/Streptomycin (10000 U/mL)<br />
*28.2mL Sterile ddH2O<br />
:-> Total 150mL, filter. <br />
:-> Chill on ice before use.<br />
<br />
=== High-Serum AMEM (HS-AMEM) ===<br />
*125mL Minimum essential medium (MEM)<br />
*20mL Heat-inactivated fetal bovine serum (56 degree, 30 min)<br />
*2mL Insulin (1 mg/mL)<br />
*2mL Glutamine (200mM)<br />
*2mL Penicillin/Streptomycin (10000 U/mL)<br />
*49mL Sterile ddH2O<br />
:-> Total 200mL, filter. <br />
:-> Chill on ice before use.<br />
<br />
= Liberase TM cell dissociation on amphibian limb tissue =<br />
----<br />
*Collect the limb tissue to dissociate into a new 100mm dish.<br />
*Wash the tissue in a 60mm dish with 0.7x PBS to remove blood and debris.<br />
*Transfer the tissue into a new 60mm dish with fresh 0.7x PBS.<br />
*Remove the skin.<br />
*Transfer the limb mesenchyme into a new 60mm dish. Remove as much liquid as possible.<br />
*Chop the tissue into <500mm^3 cubes with a #21 scalpel.<br />
*Transfer the tissue pieces into the Liberase TM solution.<br />
*Rotate on a wheel 40-50 minutes, room temperature. Vigorously shake every 15-20 minutes to disperse the tissue.<br />
**The tissue will start to clump together after ~20 minutes.<br />
*Put the tube on a rack and wait until the remaining tissue pieces to settle down.<br />
*Collect the cleared, top 3mL solution and filter through a 30mm filter cup on a 15mL falcon tube.<br />
*Mechanically dissociate the remaining tissue by pipetting the remaining 1mL solution 15-20 times with a P1000 tip.<br />
*Filter the remaining solution with the same 30mm filter cup.<br />
*Stop the enzymatic reaction by washing the FACS tube with 4mL HS-AMEM, and then filter the HS-AMEM with the same 30mm filter cup.<br />
*Collect the remaining solution at the bottom of the filter cup into the 15mL falcon tube, gently mix.<br />
::-> If doing scRNA-seq directly, continue with [[for scRNA-seq|these steps]].<br />
*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.<br />
*Wash the cell pellet with 5mL 0.7xPBS<br />
*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.<br />
**The sample is ready for downstream applications:<br />
::-> [[for FACS]]<br />
::-> [[for cell transplantation]]</div>Tzi.linhttps://wiki.tanakalab.org/index.php/With_FACSWith FACS2022-07-18T21:07:49Z<p>Tzi.lin: /* For FACS */</p>
<hr />
<div>= For FACS =<br />
----<br />
*Finish the dissociation protocol<br />
*Resuspend in 150-200uL AMEM(0)<br />
*Send to FACS facility immediately<br />
**Book FACS machine F02 because it has both GFP and mCherry lasers.<br />
**Normal setting: 85um nozzle with 0.7x FACS Flow for frog cells; 100um nozzle with 0.7x FACS Flow for axolotl cells.<br />
*Collect the cells into a 1.5mL tube with 700uL AMEM(0) inside. Note down the FACS count.<br />
*Spin down the cells. 300xrcf, 20 degree, 5 minutes.<br />
*Resuspend in 50-100uL AMEM(0)<br />
*Count the cells<br />
<br />
*The sample is ready for downstream applications:<br />
**[[For_scRNA-seq|scRNA-seq]]<br />
**[[For_transplantation|cell transplantation]]</div>Tzi.linhttps://wiki.tanakalab.org/index.php/LiberaseTM_dissociationLiberaseTM dissociation2022-07-18T20:11:09Z<p>Tzi.lin: /* Liberase TM dissociation */</p>
<hr />
<div>= Preparing reagents =<br />
----<br />
== 0.7x PBS ==<br />
*Dilute 1x PBS to 0.7x with dH2O<br />
<br />
== Liberase TM solution ==<br />
*Thaw stock Liberase TM (100x) aliquots on ice.<br />
*30uL Stock Liberase TM<br />
*2970uL 0.7x PBS in a FACS tube<br />
-> chill on ice<br />
<br />
== AMEM(0) ==<br />
*114.3mL F12:DMEM+Glutmax<br />
*1.5mL Sodium pyruvate (100mM)<br />
*1.5mL B27 supplement<br />
*1.5mL Insulin (1 mg/mL)<br />
*1.5mL MEM Non-Essential Amino Acid (NEAA)<br />
*1.5mL Penicillin/Streptomycin (10000 U/mL)<br />
*28.2mL Sterile ddH2O<br />
-> 150mL total, filter.<br />
-> Chill on ice<br />
<br />
== High-serum AMEM (HS-AMEM) ==<br />
*125mL Minimum essential medium (MEM)<br />
*20mL Heat-inactivated fetal bovine serum (56C, 30 min)<br />
*2mL Insulin (1 mg/mL)<br />
*2mL Glutamine (200mM)<br />
*2mL Penicillin/Streptomycin (10000 U/mL)<br />
*49mL Sterile ddH2O<br />
-> 200mL total, filter.<br />
-> Chill on ice<br />
<br />
= Liberase TM dissociation =<br />
'''Dissociation of Frog Limb Bud Cells''' Timing: 2-3 hours<br />
----<br />
<br />
This step describes the Liberase-based cell dissociation method to obtain high quality cell suspension for downstream applications. We recommend performing the following steps in a sterile environment.<br />
<br />
*Anaesthetise the animals and collect the limb bud tissue of your desired stages in a new 100mm dish.<br />
*Transfer the limb bud tissue into a new 60mm dish with fresh 0.7x PBS.<br />
*Remove the skin using a pair of autoclaved fine tweezers.<br />
*Transfer the limb bud mesenchyme into a new 60mm dish and remove as much liquid as possible.<br />
*Chop the limb bud mesenchyme into <500um^3 cubes.<br />
*Transfer the limb bud pieces into Liberase TM solution.<br />
*40-50 minutes on the wheel, room temperature. <br />
**The tissue should "stick" together after 20 minutes. Shake every 12-20 minutes to disperse the tissue pieces.<br />
*Put the tube on a rack to let the remaining tissue pieces to settle down at the bottom.<br />
*Transfer the clear 2mL solution on the top to a 30um filter cup on a 15mL falcon tube.<br />
*Mechanically dissociate the remaining tissue by pipetting the remaining 1mL solution with P1000 tip 15-20 times.<br />
*Transfer the solution to the same filter cup.<br />
*To stop the reaction, wash the digestion tube with 3mL HS-AMEM and transfer the medium to the same filter.<br />
**Collect the filtered solution from the bottom of the filter.<br />
*Spin down the cells, 300x rcf, 20 degree, 5 minutes.<br />
*Remove supernatant and wash with 5mL 0.7xPBS.<br />
*Spin down the cells, 300x rcf, 20 degree, 5 minutes.<br />
*The sample is ready for downstream applications.<br />
**[[ELISA]]<br />
**[[For_scRNA-seq|scRNA-seq]]<br />
**[[For_transplantation|Cell transplantation]]</div>Tzi.lin