SiRNA knock down - HuH7 cells - 24 well plate - analysis by fluorescence microscopy (Thiele lab)
From LipidomicsWiki
siRNA transfection – HuH7 cell line – 24 well plate – analysis by fluorescence microscopy
This reverse genetic approach is used to knock down specific genes followed by analysis of the resulting phenotype. The analysis of the neutral lipids stored in lipid droplets is done by fluorescent light microscopy.
The HuH7 cell line can be transfected under serum conditions by using two different reagents
Transfection reagent 1: Hiperfect, Invitrogen
siRNA stock: 2 µM
Step 1:
Plate the cells in 400 µl complete culture medium at least one day before the transfection in a 24 well plate with one coverslip in each well.
Step 2:
Day of transfection: cells should be 15 % confluent.
Preparation of the transfection solution per well:
• Mix 7 µl of siRNA stock with 100 µl Opti-MEM
• Add 6 µl Hiperfect
• Mix
• Incubate 5 min
• Add 100 µl of the transfection solution to the well
• Total volume in the well: 500 µl
Step 3:
Incubate the plate in a cell culture incubator for 24-96 hours. The incubation time depends on the target protein and the question under study.
Step 4:
Optional: 16 hours before fixation the medium conditions can be changed to oleate feeding conditions or delipidated conditions (see Preparation of delipidated FCS (Thiele lab)) .
Step 5:
Check knock-down efficiency on a visual level by checking the positive transfection controls. Fr this, it is advisable to use an siRNA targeting the mitotic kinase eg-5. Knock-down of eg-5 efficiently leads to mitotic arrest and cell death. If more than 85 % of the cells are dead (or missing), continue with step 6.
Transfection reagent 2: Interferin, BioMol
siRNA stock: 125 nM
Step 1:
Plate the cells in 500 µl complete culture medium at least one day before the transfection in a 24 well plate with one coverslip in each well.
Step 2:
Day of transfection: cells should be 15 % confluent.
Preparation of the transfection solution per well:
• Mix 7 µl of siRNA stock with 100 µl Opti-MEM
• Add 2 µl Hiperfect
• Mix
• Incubate 10 min
• Add 100 µl of the transfection solution to the well
• Total volume in the well: 500 µl
Step 3:
Incubate the plate in a cell culture incubator for 24-96 hours. The incubation time depends on the target protein and the question under study.
Step 4:
Optional: 16 hours before fixation the medium conditions can be changed to oleate feeding conditions or delipidated conditions (see Preparation of delipidated FCS (Thiele lab)) .
Step 5:
Check knock-down efficiency on a visual level by checking the positive transfection controls. Fr this, it is advisable to use an siRNA targeting the mitotic kinase eg-5. Knock-down of eg-5 efficiently leads to mitotic arrest and cell death. If more than 85 % of the cells are dead (or missing), continue with step 6.
Step 6:
Preparation for image acquisition.
Cells should be 70 – 80 % confluent.
- Remove culture medium
- Wash cells once in 1x D-PBS
- Fix cells in 1x D-PBS + 5 % Formaldehyde
- Incubate 30 – 60 min
- Wash cells three times in 1 x D-PBS
o Stain cells with staining solution by adding 200 µl/well:
o DAPI 1 µg/ml
o BODIPY493/503 1 – 2.5 µg/ml
o In 1x D-PBS
- Incubate 30 – 60 min light protected under agitation
- Briefly wash the cells three times in 1x D-PBS
- Wash the cells 3x 10 min in 1x D-PBS
- Briefly wash the cells once in water
- Mount the cells on an object slides:
o Put 5 µl Mowiol on the object slide
o Take the coverslip
o Put on without airbubbles
Store slides in the dark in the fridge until image acquisition.
