The WEHI 231 cells were transfected with NS5A-mCherry. 12 hours after stimulation cells were stimulated with anti-IgM. At T=36 cells were collected and stained with either ER tracker or Bodipy. Cells were then prepared for flow cytometry
The WEHI 231 cells were transfected with NS5A-mCherry (100 uL). A control of Thapsigargin was used, cells in this group were treated with 87.5 (0.250mM) Thapsigargin for 4 hours before RNA isolation.
T=0 cells were collected an hour after stimulation. Cells were lysed with 500uL TRIzole reagent and frozen at -80C
Cells were stimulated with 35 uL of anti-IgM 12 hours after transfection.
T=36 cells were collected and lysed with 1 mL TRIzole reagent.
RNA from both the T=0 and T=36 samples was isolated according to TRIzole manufacturing protocol.
Nanodrop concentrations were as follows:
T=0 mock 225.5 ng/uL
T= mock stim 1956.5 ng/uL
T=0 NS5A 575 ng/uL
T=0 NS5A stim 1487 ng/uL
T=0 Thap 844.5 ng/uL
T=36 mock 24322 ng/uL
T=36 mock stim 400 ng/uL
T=36 NS5A 4415 ng/uL
T=36 NS5A stim 226 ng/uL
T=36 Thap 417.5 ng/uL
The integrity of the RNA was determined using a 1% agarose gel results to follow
Cells from experiments that are to be analyzed via Flow Cytometry or Electron Microscopy are prepared in the following manner:
Harvest cells and centrifuge (1000 rpm, 3 min), vortex the cell pellet with .5mL of cold PBS. Add another .5mL of cold PBS and completely resuspend the pellet. Centrifuge the cells again. Vortex the cell pellet was with .2mL of cold PBS and add the cells dropwise to .750mL of cold 75% ethanol. Incubate the cells on ice for 30 minutes before being transferring to the fridge. Cells can be stored in this fashion for up to 1 week.
Harvest 1.5 mL of cells and centrifuge off media (1000 rpm, 5 min). Wash twice with 500 uL of PBS. Fix cells with 150 uL of either 2% paraformaldehyde or cold 75% ethanol for 10 minutes in the fridge. Resuspend cell pellet in PBS.
WEHI 231 cells were transfected with NS5A-mCherry according to this protocol:
Project: Regulation of Phosphotidylinositol-4-phosphate by Ttc7 and its role in plasma cell differentiation and autoantibody production
Purpose: To transfect WEHI 231 cells with the Hepatitis C protein NS5A-mCherry.
Experimental Design: WEHI 231 cells were transfected using the BTX T820 Electrosquareporator. See below for protocol
Materials: 15 mL conical tube, transfection buffer, WEHI 231 media, 6 well culture plate, WEHI 231 cells, centrifuge, transfection cuvettes.
1. To start obtain a cell count, for this transfection 6 million cells were needed. Spin down the necessary volume of cells and wash twice with 5 mL of 1X PBS (for cell culture).
2. Resuspend cells in transfection buffer, each transfection should be resuspended in 1.5mL of transfection buffer. 3 mL of transfection buffer is needed (1.5 for mock transfection, 1.5 for NS5A-mCherry). Aliquot the 1.5 mL of cells resuspended in transfection buffer to 1.5mL eppindorf tubes.
3. Plasmid preparation- at this step plasmid will be added to the eppindorf tube of cells. For 6 million cells, 2 ug of plasmid is needed. To calculate the volume of plasmid needed: NS5A-mCherry concentration is 296ng/uL. (2X10-6g/1)(1/296X10-9g)= 6.75 uL At this point add the necessary volume of plasmid to the respective eppindorf tubes.
*double the amount of plasmid was used*
4. Transfer contents of eppindorf tubes to respective cuvettes. Immediately put cells on ice and electroporate. WEHI 231 cells are electroporated at the following settings: 1 pulse, low voltage (LV), 50msec, 170V. After electroporation return to ice and bring back to culture hood.
5. The electroporation leaves a white lipid residue at the top of each cuvette, remove this or avoid it when transferring the contents of the cuvettes to the 6 well plate. Add 1.5 mL of media to each well. Allow cells to incubate and recover for 24 hours.
6. After 24 hours stimulate the cells with anti-IgM for one hour.
7. To harvest cells transfer 1.5mL of the contents of each well to an eppindorf tube (this half of the procedure is not done under sterile conditions). Centrifuge cells at 1000 g for 5 min. While cells are centrifuging dilute both ER Tracker and Bodipy in Hanks HBBS (different solutions) by adding 1 uL of dye to 1 mL of Hanks. Stain for 20 minutes, centrifuge cells to remove stain. Wash twice with 500 uL of PBS.
*This dye dilution results in a 1 ug/mL concentration*
8. To fix cells add 150 uL of 2% Paraformaldehyde. Cover and let sit in the fridge for 10 min. Centrifuge PF off of the cells. Turn centrifuge up to 1500 g, fixed cells need a little higher g. Pour of PF in designated waste. Wash cells with 500 uL PBS.
9. Stain cells with DAPI. DAPI stock comes at 1 mg/mL. 1 ug/mL is necessary for cell staining. To dilute DAPI add 1 uL of DAPI to 1 mL of PBS. Add 200 uL of 1 ug/mL DAPI to each eppindorf tube. Cover and let sit for 20 min.
10. Centrifuge off DAPI stain, leave approx. 50 uL to resuspend cells in. 10 uL of cells will be used to mount on slide.
11. Add one drop (approx. 10 uL) of Molecular Probes ProLong Gold Antifade reagent to each well on slide. Add 10 uL of cells to each well. Add coverslip, secure in place with clear nail polish. Let slides sit overnight. It most likley will take about a week for the cells to completely set.
12. Use confocal microscope to view cells.
To come, the computer with the confocal imaging software currently has a virus. Images to come!
To resuspend cells it is best to either flick the tubes to resuspend the pellet, or run the tube up and down a tube holder rather than using a pipetter. This prevents tearing of the cells.
This experiment has been repeated for a total of 3 times.
Sample Storage Location:
In slide box in Michaela Sangillos lab drawer.
This experiment will be repeated again tomorrow. At T=0 and T=36 300 uL of cells will be harvested for flow cytometry.
WEHI 231 cells were transfected with NS5A-mCherry. The plasmid used came from the last round of propagation so 25 uL had to be used. It may be necessary to use 50 uL depending on outcome of this experiment. A 24 well plate was used instead of a 6 well plate. This transfection will be stimulated for 1 hour with anti-IgM after 24 hours. 750 uL of transfected cells was added to 750 uL of media in each well.
NS5A was purified from Top10 E. coli. The concentration was not desirable. The protocol will be repeated with more time for the E. coli to reach mid log phase growth.
The experiment was repeated, started 11/12/14. At T=0 andT=36 300uL of cells were harvested and prepared for electron microscopy.
The dual transfection was repeated a third time.
Today the dual transfection of WEHI 231 cells with NS5A-mCherry and PI4K-GFP was repeated.