Daniel:Notebook/ComboLock/2016-7-22: Difference between revisions
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=Cell Retention Test= | =Cell Retention Test= | ||
Revision as of 20:53, 22 July 2016
Cell Retention Test
I've been worried about cell retention during the spins for some time, and I've been thinking about how to test it. So, I'm going to run through the protocol (without probes) but using hematoxylin to stain the cells.
Buffers
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
PBS | 10X | 1X | 10 | 100 uL |
SSC | 20X | 4X | 5 | 200 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
nf H2O | NA | NA | NA | 700 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 100 uL |
SSC | 20X | 1X | 20 | 50 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
PBS | 10X | 1X | 10 | 100 uL |
nf H2O | NA | NA | NA | 850 uL |
Protocol
- C-Probe Hybridization
- Measure cell concentrations in cell counter (Human - 400,000 cells/mL)
- Add cells to a new tube such that there are 200,000 cells present (500 uL)
- Pellet cells via centrifugation at 600g for 3 min
- Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
- Wash 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Resuspend cells in 100 uL CPH buffer
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 100 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 100 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend pellet in 100 uL LPH buffer
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 100 uL wash buffer pelleting cells at 1000xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
This is the last step where you change buffer in the protocol. If I still have cells, we're good.
Results
I still had cells at this point, but the hematoxylin really helps visualize the cell pellet and doesn't interfere with the chemistry (so it has been reported) so I think that it is a good idea to use it.
Latch 100 and Dextran Test
Buffer Prep
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 5 mL (uL) |
PBS | 10X | 1X | 10 | 500 uL |
SSC | 20X | 4X | 5 | 1000 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 5 uL |
nf H2O | NA | NA | NA | 4.5 mL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 100 uL |
SSC | 20X | 1X | 20 | 50 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
PBS | 10X | 1X | 10 | 100 uL |
nf H2O | NA | NA | NA | 850 uL |
CPH Buffer with 10% Dextran Sulfate
Reagent | Stock Conc. | Final Conc. | Amt. Added |
SSC | 20X | 1X | 50 uL |
Tween 20 | 10% | 0.10% | 10 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1 uL |
Riboside Vanadyl Complex | 200 mM | 20 mM | 100 uL |
Poly Vinylsulfonic Acid | 25% | 2.50% | 100 uL |
Salmon Sperm | 10 mg/mL | 100 ug/mL | 10 uL |
Dextran Sulfate | 25% | 10% | 400 uL |
Probes | 100 uM | 100 nM | 1 uL/probe |
Water | NA | NA | 340 uL |
Total | 1 mL |
Protocol
- C-Probe Hybridization
- Measure cell concentrations in cell counter
- Human - cells/mL; Mouse - cells/mL
- Add cells to a new tube such that there are 100000 cells present
- Pellet cells via centrifugation at 600g for 3 min
- Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
- Wash 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Human- uL; Mouse- uL
- Pellet cells via centrifugation at 600g for 3 min
- Combine 17 uL of each C probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer according to sample matrix
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
C Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | U87MG | 3T3 Cells | Treatment | |
Sample 1 | X | X | X | None | |
Sample 2 | X | X | X | Dextran | |
Sample 3 | X | X | X | 10X Latch | |
Sample 4 | X | None | |||
Sample 5 | X | X | X | None | |
Sample 6 | X | X | None |