The dual transfection was repeated a third time.
Today the dual transfection of WEHI 231 cells with NS5A-mCherry and PI4K-GFP was repeated.
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 PI4K-GFP and NS5A-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 NS5A-mCherry/PI4K-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: PI4K-GFP concentration is 192.9ng/uL. (2X10-6g/1)(1/192.9X10-9g)= 10.4 uL. Optimization experiments found that 14 uL of the plasmid forNS5A-mCherry should be tranfected. 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.
Lupus is a chronic autoimmune disease where the immune defence system attacks the cells, tissues, and organ systems rather than protecting the body against harmful pathogens, such as viruses and bacteria. More than 1.5 million Americans have lupus and over 90% are female, and women of color have two to three times higher risk of having Lupus. Antibodies produced by the immune system attack the skin, kidneys, heart, blood, brain, joints, and lungs and which is the leading cause of early onset kidney disease, cardiovascular disease, and stroke in women. The key to better health is the early detection of Lupus and therapeutic management; however Lupus is difficult to diagnose and often goes undiagnosed or misdiagnosed, sometimes for years.
To better understand Lupus it is important to characterize the process of how and why the immune system attacks its own body. The development of the immune system has several checks and balances to insure it is not harmful to its own body while protecting against harmful microorganisms. Antibodies are produced to detect and remove these harmful microorganisms. So why then do patients with Lupus produce antibodies that attack their own bodies? This is the key question that will be addressed by this project. A new idea will be explored that links a specific type of cellular stress with the uncoupling of the normal controls of the B lymphocytes—the cells that produce antibodies—and lead to the production of autoantibodies, the antibodies that attack one’s own body. Specific components of the cell membrane are required to maintain the normal function of the cells, this research will focus on one type of membrane component, phosphatidylinositol-4phosphate (PI4P for short), and the enzyme that produces PI4P and determine if defects in the regulation of this system are associated with inducing stress in B lymphocytes and allowing autoantibodies to be produced.
The project will use B Lymphocyte cell lines that can be easily cultured as a model of B lymphocytes in the body. These cell lines will be manipulated to alter the regulation of one of the processes associated with the production of PI4P and measure 1) the level of a specific type of cellular stress and 2) the ability of these cells to produce antibodies. This research will provide insight into a new model of autoantibody production and with a more detailed understanding of this process it is hoped that new diagnostic tools or therapies can be developed based on these findings.