Transfection of Target Cells with dCas9 and sgRNA Expression Vectors
One day prior to transfection, seed 12-well cell culture plates with 6x104 HEK293T cells in 1 mL DMEM/GlutaMAX supplemented with 10% FBS per well. Incubate at 37°C and 5% CO2.
Twenty-four hours after plating cells, prepare the transfection complexes, scaling up the mixture masses and volumes as needed:
i. For each well, combine the following expression vector DNAs.
250 ng pPB: pCAG-GID1-VPR-IRES-Puro-WPRE pPGK-GAI-tagBFP-Sp dCas9
250 ng pPB: pCAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA pPGK-ABI-tagBFP-Sa dCas9
83.3 ng pHR: pU6-Sp sgCD95-1 pCMV-EGFP
83.3 ng pHR: pU6-Sp sgCD95-2 pCMV-EGFP
83.3 ng pHR: pU6-Sp sgCD95-3 pCMV-EGFP
83.3 ng pHR: pU6-Sa sgv2CXCR4-1 pCMV-EGFP
83.3 ng pHR: pU6-Sa sgv2CXCR4-2 pCMV-EGFP
83.3 ng pHR: pU6-Sa sgv2CXCR4-3 pCMV-EGFP
ii. Add this 1 μg DNA mixture to 100 μL Opti-MEM Reduced-Serum Medium in a microcentrifuge tube. Mix well by pipetting up and down.
iii. Add 3 μL Mirus TransIT-LT1 Transfection Reagent into the same tube. Mix well by pipetting up and down.
iv. Allow transfection complexes to form for 30 min at room temperature.
- Add 100 μL of the transfection mixture to each well of the 12-well plate containing plated HEK293T cells. Mix well by rocking the plate back and forth. Incubate 24 h at 37°C and 5% CO2.
Induction of Target Cells with Abscisic acid/Gibberellin
- Twenty-four hours after transfection, prepare small molecules for induction as follows:
Prepare 500x concentrated stocks of abscisic acid and gibberellin.
Abscisic acid: Dissolve 13.22 mg abscisic acid in 1 mL DMSO to make a 50 mM solution.
Gibberellin: Dissolve 2.09 mg gibberellic acid acetyoxymethyl ester in 1 mL DMSO to make a 5 mM solution.
These stocks can be stored at -20°C.
Add 2 μL of the 500x concentrated stocks of abscisic acid and/or gibberellin to each well of the 12-well plate containing transfected HEK293T cells.
Add DMSO in parallel such that 4 μL total volume of (DMSO + abscisic acid + gibberellin) are added to each well. Mix well by rocking the plate back and forth. Incubate 48 h at 37°C and 5% CO2.
Immunostaining for CD95/CXCR4
- Forty-eight hours after induction, harvest cells as follows:
i. Aspirate cell culture media from each well.
ii. Add 300 μL Versene solution per well, and incubate 5 min at 37°C and 5% CO2 to dissociate cells.
Transfer Versene solution containing dissociated cells into one microfuge tube per well. Centrifuge 2 min at 3,500 RPM in a microcentrifuge.
Prepare a blocking buffer containing 10% FBS in DPBS.
Aspirate Versene solution from pelleted cells, taking care not to disturb the cell pellet. Resuspend cells in 375 μL blocking buffer by pipetting up and down. Centrifuge 2 min at 3,500 RPM.
Prepare staining solutions as follows, scaling up volumes as needed:
For each sample:
Add 0.2 μL APC-CXCR4 antibody (1:500, final concentration 0.4 μg/mL) and 0.5 μL PE-CD95 antibody (1:200, final concentration 0.5 μg/mL) to 100 μL 10% FBS in PBS.
For isotype controls:
Add 0.2 μL APC IgG2 antibody (1:500, final concentration 0.4 μg/mL) or 0.25 μL PE IgG1 antibody (1:400, final concentration 0.5 μg/mL) to 100 μL 10% FBS in PBS.
Aspirate blocking buffer from pelleted cells, taking care not to disturb the cell pellet. Resuspend cells in 100 μL staining solution by pipetting up and down.
Incubate cells 1 h at room temperature protected from light. Centrifuge 2 min at 3,500 RPM.
Aspirate staining solution from pelleted cells, taking care not to disturb the cell pellet. Resuspend cells in 375 μL blocking buffer by pipetting up and down. Centrifuge 2 min at 3,500 RPM.
Resuspend cells in 150 μL blocking buffer and immediately analyze by flow cytometry.