Lipofectamine Transient Transfection of COS7 cells to analyze by
Immuno-Fluorescense (3/1/00 BLJ/CMS)
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Experiment Purpose and Cell plate containers used:
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Day One, Seed cells on Coverslips:
Prepare 6 well plates by adding Baked/Sterile 22x22 (1.5 thickness) coverslips to wells.
Add 2 mls of media to each well and Seed with 400,000 COS7 cells per Coverslip. Make
sure and evenly distribute the cells on the Coverslips by swirling plate. Let cells attach
by incubating at 37˚C overnight.
Day Two, Transient Transfect Cells:
For all Transfection of multiple wells in 6 well plate, Multiply cDNA, Opti-MEM, and
Lipofectamine quantity used by number of wells to transfect.
A) For each well of a 6 well plate: in 1st polystyrene Tube (Falcon cat# 2057), add 1µg
of cDNA to 100µl of Opti-MEM (Gibco Cat#31985). If Co-transfecting; add second
1µg sample of cDNA directley to same sample.
B) For each well of a 6 well plate: in 2nd polystyrene tube add 8µl of Lipofectamine
(Life-technologies Cat# 18324-012) and 100µl of Opti-MEM.
C) Let lipid and cDNA incubate at R.T. in Hood for 5 minutes until proceeding.
D) Add 108µl lipofectamine sample to cDNA sample (always add Lipid to cDNA), mix
sample by GENTLY swirling, and let incubate at R.T. in hood for 30minutes.
E) At ~ 25 minutes wash Cos7 Cells in 6 well plate x2 with Opti-MEM. Remove 2nd
wash and add back 1ml of Opti-MEM to each well.
F) After 30 minute incubation of Lipid and cDNA, add 208µl sample of lipid-cDNA
(from step d) complexes to each coverslip in 6well plate. Make sure and evenly
distribute sample by GENTLY swirlling.
G) Let cells/cDNA-lipid complexes incubate for 5 hours at 37˚C.
H) After 5 hours remove 6 well plate and wash 2X with Opti-MEM, Wash once with
COS7 complete media and replace with 2ml/well of COS7 complete media.
I) Let cells incubate for 48 hours at 37˚C until experimentally analyzing, feeding every
day with Complete media.
Day Four, Stain Coverslips for Confocal/ Epifluorescence viewing:
MDCK CAL/ER/ERGIC/-Adaptin IF Staining Protocol (2/18/00 BLJ)
Modified from 2/1/00 protocol to better preserve internal structure of cells by reducing
severity of permeablization and fixation.
For 6 well plates, all wash volumes are 2 mL (DO NOT SHAKE)
1)Wash cells in PBSgs++ to remove medium (30 s RT)
2)Fix in 2% paraformaldehyde 5% Sucrose in PBS++ (20 min at RT)
3)Wash with PBSGS++ (2x 5min at RT)
4)Quench paraformaldehyde with 100 mM glycine in PBS++ (10 min at RT)
4a)To stop, replace glycine solution with PBSgs++ wrap in plastic then foil and store at
4ºC.
If you are going to store in 4C for a long time add Na azide, store in 0.02% Na
Azide/PBS++.
5)Wash with Permeablization Buffer (1x 5min at RT)
6)Wash with PBSgs++ (3x 5min at RT)
7)Block with GSDB (30 min at RT)
Avoid exposing slips to light from this point on.
8)Incubate with antibody in GSDB 100 µL/slide on Parafilm, cover (1 hr at RT)
Cup parafilm to size of dish. Drop 100µl of Ab/GSDB to appropriate labeled grid area.
Place C.S. cell side down on the bead (no air bubbles.)
9)Wash with GSDB (3x 5 min at RT)
10)Incubate with Goat Anti-Rabbit Texas Red 1:100 in GSDB 100 µL/Slide on Parafilm
(1 hr at RT)
11).Wash with PBSgs++ (4x 5 min)
12).Mount with Prolong™ anti-fading agent. Do not Velap slides.
A) 1 ml of reagen B (no bubbles)(do not push out all fluid). Add to reagent A.
Solubilize carefully, by pipetting.
B) Sonicate if possible
C) Add 15µl of solution to coverslip, where slide will be added.
D) Put C.S. on kemwibe by edges (drop fluid), place onto slide over prolong. Cell Side
Down.
E) But in box with drying agent/ in dark over night.
PBS++
989 mL PBS
10 mL 100 mM MgCl2
1 mL 100 mM CaCl2 dropwise while stirring
PBSGS++ (~120 mL per six-well plate)
120 mL PBS++
1.2 mL Goat Serum
Permeabilization Buffer (12 mL per six-well plate)
For 12 mL
For 36 mL
Comp
Triton X-100 12 µL
36 µL
0.1%
Goat Serum 120 µL
360 µL
1%
PBS++
12 mL
36 mL
Prepare in 50 mL conical vial put on shaker to solublize Triton X-100. Prepare in
advance.
GSDB (~50 mL per six-well plate)
For 50 mL
Comp
PBS++
42.5 mL
Goat Serum
7.5 mL
15%
2% Paraformaldehyde 5% Sucrose (12 ml per six-well plate)
For 12 mL
For 50 mL
Comp
Paraformaldehyde
240 µL
1 mL
2%
Sucrose
0.6 g
2.5 g
5%
PBS++
12 mL
45 mL
PROLONG ANTIFADE: Mix Gently and cetrifuge to remove large air bubbles,
sonicate Prolong to better dissolve powdered reagent and to remove small residual air
bubbles.