Category Archives: Michaela Sangillo

WEHI 231 Transfection with EFR3b-GFP and EFR3b-mCherry

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 EFR3b-GFP and EFR3b-mCherry. To establish repeatable transfection 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. 4.5 mL of transfection buffer is needed (1.5 for mock transfection, 1.5 for EFR3b-GFP transfection, 1.5 for EFR3b-mCherry 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: EFR3b-GFP concentration is  120.1ng/uL. (2X10-6g/1)(1/120.1X10-9g)= 16.65uL. 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. 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. Wash twice with 500 uL of PBS.

7. 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.

8. 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.

 

Results:

To come, the computer with the confocal imaging software currently has a virus. Images to come!

Notes:

To be clear these are separate transfections per each vector. Not a dual transfection.

Sample Storage Location:

In slide box in Michaela Sangillos lab drawer.

File Locations:

Tba

WEHI 231 Transfection with GFP and mCherry control vectors

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 GFP and mCherry control vectors. To establish repeatable transfection 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. 4.5 mL of transfection buffer is needed (1.5 for mock transfection, 1.5 for GFP transfection, 1.5 for mCherry 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, 1.5 ug of plasmid is needed. To calculate the volume of plasmid needed: GFP concentration is 104.6 ng/uL. (1.5X10-6g/1)(1/104.6X10-9g)= 14.34uL. mCherry concentration is 135.6ng/uL. (1.5X10-6g/1)(1/135.6X10-9g)= 11.06 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. 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. Wash twice with 500 uL of PBS.

7. 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.

8. 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.

 

Results:

To come, the computer with the confocal imaging software currently has a virus. Images to come!

Notes:

The amount of plasmid was changed to 2 ug of plasmid per 6 million cells after this transfection.

Sample Storage Location:

In slide box in Michaela Sangillos lab drawer.

File Locations:

Tba

Cell Mask Staining

Project: 

 

Purpose:

To stain plasma membrane and use a live stain for nucleus

Experimental Design:

 

To prepare Cell Mask:
Official Protocol calls for 1 mL of stain per coverslip- needs to be diluted.
Dilution:
Stock= 5 mg/mL
Want 2.5 ug/mL
Add 2.5 uL of stain to 5 mL of PBS
Stain cells with Cell Mask for 5 min.

To prepare Hoescht:
Want 1 ug/mL in PBS
Stock= 5 ug/mL
Add 1 uL of stain to 5 mL of PBS
Stain cells with Hoescht for 30 min according to Pelsue, stained for 5 min this time

Harvest Cells- took 5 mL of cells
Add stain to cells + media
Spin off stain
Wash with PBS
Transfer to eppindorf tubes
Fix cells

Do PBS wash twice
Pour off all but approx. 50 uL of PBS and flick to resuspend
Add 150 uL of Paraformaldehyde (2-4% for WEHI)
Put in fridge under foil for 10 minutes
Spin at 1500G to 2000G for 3 minutes and check formation of pellet- spin more accordingly
Remove Paraformaldehyde (put in its own waste)
Add 200 uL of PBS
Spin (still at higher G) for 5 minutes
Pour off all but approx. 50 uL again and flick to resuspend
Use 1 drop Promega gold mounting medium (1 drop=10uL)
Add 10 uL of cells to well with mounting medium in it
Mix and spread around well
Add coverslip and nail polish it down
Allow to set- check in 2 days and again in 6

Results:

Both the plasma membrane and nucleus are stained.

Notes:

Important to do cell count beforehand

Sample Storage Location:

 

File Locations: