Hi Everyone! I am looking forward to the CRISPR event this Thursday! 

The startup "FIND Genomics" will be present! We are developing an enabling technology to allow innovation in cell biology and CRISPR to thrive. 

Heads-up: **we are looking to hire a full time cell expert, project manager for our NIH-SBIR project** -- check out the job posting here: https://www.linkedin.com/jobs/view/2920493708

If you are a cell biologist (PhD, Postdoc), passionate about cell biology but also curious to see "what's out there"  -- come and chat with us! 

I hope to meet many of you this Thursday! 

Best, Sophie

(We are also looking for (part-time) interns!)



Job posting for cell biologist interested to broaden their horizons!

Pilot experiment to determine general translation effects and optimal conditions for electroporation of TVA cell line.

- Renilla luciferase shows cap dependent translation 
- Firefly luciferase shows cap independent translation 
- Used Promega Dual-Luciferase Reporter Assay System and FB12 Luminometer from Berthold

 Protocol 

1. Warm 5 ml media in 6 well plate 

2. Resuspend cells in 400 ul serum free media. Use 3 million cells per condition. 

3. Add 1 ug pRSTF-CVB3 plasmid per 1 million cells. 

4. Electroporate cells in 0.4 cm cuvette electroporator 

5. Slowly add the 400 ul of electroporated cells to the warm 5 ml of media. 

6. Incubate the cells in media for 2 hours to let them recover. 

7. Aliquot 1ml of cell suspension per well and add the drug treatment. 

8. Incubate for 16 hours. 

9. Spin down cells (500 G, 5 min).  

10. Wash with PBS and spin down again. 

11. Resuspend in 25 ul passive lysis buffer (samples at this point can be stored in the -80 for a long period of time). 

12. Lyse by freezing cells in -80 and thawing, 5 minutes each. Do 2 freeze/thaw cycles. 

13. Combine 10 ul lysate and 50 ul LAR2 substrate and measure in luminometer to read Firefly luciferase (cap independent translation). 

14. Immediately add 50 ul Stop and Glow solution and measure in luminometer to read Renilla luciferase (cap dependent translation). 

15. Compare ratio of Renilla to Firefly luciferase for readouts. 

 

TVA Electroporation Optimization  

8 conditions, 3 million cells each (24 million total) 

Each condition is with or without pRSTF-CVB3 in separate 0.4 cm cuvettes 

250 uF 250 V 
250 uF 300 V 
975 uF 250 V 
975 uF 300 V 

Results 

All luciferase values too low, likely no transformation 

TVA Cell Line Electroporation Optimization

DNA stains enable a commonly used FACS method of monitoring a compound’s effects on cell cycle progression. 


1. Transfer cells from culture dishes to FACS tubes. Make sure you pipet vigorously to get all the lightly adherent cells. The protocol assume 0.5 million cells per sample, so scale up or down with the volumes for your samples. 

2. If needed, stain for surface antigens.

3. Spin cells for 5 minutes, 500 g in a microcentrifuge. Discard supernatants afterwards.

4. Resuspend the cell pellet in 250 uL wash buffer (1x PBS with 5mM EDTA). 

5. Add 125uL 100% COLD ethanol and vortex immediately. Repeat once. 

6. Incubate at room temperature for at least 30 minutes. This cell suspension can be left at 4 C overnight instead if necessary.

7. Spin cells at 5 min, 500 g. Discard supernatants afterwards.

8. Add 250 uL propidium iodide solution (0.1 mg/ml propidium iodide in wash buffer). Place samples on ice.

9. The exact parameters and protocol at this point are dependent on your flow cytometer, but in general start with gating out cell debris using SSC/FSC and doublets using FL2-H/FL2-W. PI has an excitation wavelength of 488nm and maximal emission at 617nm.

10. Collect at around 10,000 events at low speed settings using cell count vs FL2-H as the primary indicator of cell cycle. There will be 2 primary peaks representing G0/G1 and G2 phase respectively, with the G2 phase having double the FL2 height due to having twice the amount of DNA. Cells with less FL2 height than the G1 population are sub-G1 (dying) and cells in between the two main peaks are in the S phase.

Cell Cycle Progression Analysis

A commonly used quantifiable metric for protein phosphorylation is achieved through the use of fluorescence tagged antibodies specific for phosphorylated protein sites. Post treatment cell samples incubated with these antibodies can be analyzed via flow cytometry to measure their respective levels of phosphorylation. Be sure to include unstained controls and secondary antibody only controls if necessary.


1. Treat 0.5-2x106 cells per condition for 2-3 hours.

2. Collect cells to conical tubes / eppitubes and spin down cells (500g, 5 minutes).

3. Prechill 810µl Methanol in -20 C freezer.

4. Resuspend pellet in 100µl PBS (does not have to be sterile).

5. Add 100µl of 4% paraformaldehyde (PFA)  -or-  25µl of 16% PFA to each sample.

6. Incubate 10 min at room temperature.

7. Add 800µl of ice cold methanol 100% methanol. At this stage, you can store at -20oC indefinitely or incubate samples for 30 minutes before proceeding to the next step.

8. Spin down cells 12,000rpm, 5min. This spin should be as fast as possible to ensure maximum sample collection.

9. Add 1 ml PBS to each sample.

10. Spin down cells 12,000rpm, 5min.

11. Prepare 100 uL of primary antibody master mix per sample. Concentrations may need to be 2-5 times higher than an equivalent western blot procedure.

12. Discard sample supernatant and add 100µl of diluted antibody in PBS. 

13. Incubate 30 minutes in the dark on ice (or a fridge).

14. Wash by adding 1 mL PBS and incubate for 5 minutes at room temperature. Spin down cells at 8000 rpm, 5 minutes. Prepare secondary antibody solution in the meantime if necessary (100 uL solution per sample).

15. Remove supernatant. If using secondary antibody repeat resuspension, incubation, and wash steps with secondary antibody solution. If proceeding to FACS analysis, resuspend in 250 uL PBS.

16. Analyze via FACS.

Phosphoflow Protocol

Mark Wesson is a highly skilled healthcare program professional with the skills needed to create high-quality healthcare programs. He has experience leading the analysis and restructuring of programs to improve the quality of care for patients. He is also adept at cultivating innovation using the latest technologies to improve information management and communication. Throughout his career, he has found and applied new and more effective ways of ensuring high-quality patient care. 

Hi I'm Kris. I received my PhD in Medical Biology studying genome engineering with CRISPR/Cas, ZFNs, and TALENs. I have multiple peer-reviewed scientific publications and awards for my research. Over the last 8 years I've worked to develop gene therapies from HIV/AIDS to hemoglobinopathies. Please feel free to direct any gene editing or related workflow questions to me, I'm happy to help! 
www.arkitekt.bio                         
www.crisprclassroom.org

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