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 both the Ttc7-fsn-GFP and EFR3b-mCherry. To establish repeatable dual transfection and stimulation technique.
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 EFR3b-mCherry/Ttc7-wt-GFP transfection). 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: Ttc7-wt-GFP concentration is 149.5ng/uL. (2X10-6g/1)(1/149.5X10-9g)= 13.4 uL. mCherry concentration is125 ng/uL. (2X10-6g/1)(1/125X10-9g)=16 uL. At this point add the necessary volume of plasmid to the respective eppindorf tubes.
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. At this point the protocol changes slightly from the Dual transfection protocol, the next step is the stimulation. After the 24 hour incubation, transfer 1 mL of cells from the well with the dual transfection. To that well add the anti-IgM. The dose is 10 ug/mL. The concentration of anti-IgM comes at 0.5mg/mL. To calculate: 0.5mg/mL= 500ug/mL. Becomes cross multiplication[500ug/1mL]X[10ug/X]= 10ug/500ug=0.02mL=20uL. After moving 1 mL of dual transfected cells to a separate well, add 20 uL of anti-IgM to the cells for one hour. After the hour, continue the protocol as usual.
7. To harvest cells transfer 1 mL 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. Wash twice with 500 uL of PBS.
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!
The anti-IgM is Goat F(ab’)2 anti Mouse IgM from Southern Biotech
Sample Storage Location:
In slide box in Michaela Sangillos lab drawer.