E
Extraction Buffer (MTT assay; for T cell Proliferation)
5 ml H2O
5 ml DMF (dimethlyformamide)
2 g SDS
Eosin
12 g Eosin Y
3 g Phloxine B (Sigma)
QS to 500 ml with 70% EtOH
EDTA (0.5 M)(disodium ethylene diamine tetraacetate)
186 g EDTA
800 ml H2O)
20 g NaOH
Stir until all EDTA goes into solution.
Adjust pH to 8.0 w/ NaOH
QS to 1 L
Elution Buffer (1X for Nickel Columns)
100 ml 500 ml
200 mM Imidazole 1.35 g 6.8 g
0.5 M NaCl 2.93 g 14.6 g
20 mM Tris HCL pH 8.0 2 ml 1 M stock 10 ml 2 M stock
pH to 7.9
Filter, store at 4°C
Extinction Coefficient
Σ280= (5600x #W) + (1400x #Y) + (200 x #F)
ELISA (example)
1-Coat plate with 0.1 µg target antigen in 50 µl PBS o/n @ 4°C. SEE ELISA TEMPLATE (triplicates).
Single Well Template.
NOTE: Peptides may need to be coated using Sodium Bicarbonate Buffer
2-Wash 3X with PBS 0.05% Tween 20. Typically this is done with an automatic plate washer or
hand-held washer. If not available you can use a squirt bottle to flush the wells. Crude but
effective.
After each wash step, tap plates (upside down) over a paper towel to remove an remaining wash
buffer.
3-Block with 200 µl/well SuperBlock (SB) (Pierce). 10% BSA dissolved in PBS is also okay but
SuperBlock works!!!
-Make all dilutions of antibodies from this step forward in SB
(or whatever your blocking agent is.
-Vortex all antibodies or reagents added to respective wells.
4-Wash 3X with PBS 0.05% Tween 20.
5-Add 100 µl of primary antibody. Depending on what this is it can be diluted 1:100-1:10,000.
Incubate at 37°C for 1 h.
6-Wash 5X with PBS 0.05% Tween 20.
7-Add 100 µl of secondary antibody. For this example assume it is alkaline phosphatase-
conjugated. Typically this can be diluted between 1:1000 and 1:5000. Incubate at 37°C for 1 h.
8-Wash 5X with PBS 0.05% Tween 20.
9-Develop using 1 Sigma Phosphatase tablet dissolved
in DEA. 1 tablet/5 ml DEA (See DEA protocol). 100 µl/well.
Develop between 30 min up to 3 h.
10-Read O.D. @ 405 nm.
E=mc2 Explained
F
Ficoll
This protocol written in the context of 'cleaning' up dead cells following a T cell stimulation with
irradiated/mitomycin C-treated APCs+Antigen.
1-Warm Ficoll to RT.
2-Warm the centrifuge to RT and set Acceleration and Brake to Zero.
3-Pipet 3 ml Ficoll into a 15 ml conical and overlay with 5 ml of media containing cells.
4-Centrifuge for 15 min.
5-Carefully pipet out the cells at the Ficoll-media interface.
6-Wash cells at least 3X with complete CTL.
Flow Cytometry
GENERAL FLOW CYTOMETRY PROTOCOL
Materials needed
FACS Buffer
PBS
.1 % BSA
.01% NaN3
Fetal Bovine Serum (FBS)
Primary antibody (biotinylated, fluorescently labeled, or unlabeled)
Secondary antibody (biotinylated or fluorescently labeled)
3rd stage (Streptavidin –fluorescent conjugate)
For multiple color staining fluorescent tags can be: GFP, FITC or Alexa 488 (FL1)
PE (FL2)
CyChrome or PerCP (FL3)
Procedure
- Wash cells 1X with at least 4 volumes of PBS
- Resuspend cells at 1 X 10_6 cells/ml in FACS buffer, put 1 ml/tube in Fisher 5 ml polystyrene stubs
- Block the cells in FACS buffer + 10% FCS for 15-30 minutes on ice
- Add 4 ml of FACs buffer to each tube, centrifuge 5 min at 2000 rpm
- Discard supernatant
- Vortex cells (there is approximately 100µl of liquid in each tube)
- Add appropriate amount of primary antibody to each tube (100-200 µl final volume in the tube)
most Abs work around 10 µg/ml
- Vortex tubes, incubate on ice for 30 minutes COVERED
- Add Secondary antibody at appropriate dilution to the tubes (most Abs work around 10 µg/ml)
- Add 4 ml FACs buffer to each tube, centrifuge 5 min @ 2000 rpm, repeat wash 2 more times
- Discard supernatant and vortex tubes
- If necessary, add Streptavidin at 1:1000 dilution (100-200 µl final volume in the tubes)
- Incubate on ice 20 min COVERED
- Add 4 ml FACs buffer to each tube, centrifuge 5 min @ 2000 rpm, repeat wash 2 more times
- Discard supernatant and vortex tubes
- Add 400 µl FACS buffer to each tube and analyze on flow cytometer immediately or fix in
paraformaldehyde (200 µl FACS + 200 µl 2% paraformaldeyde in each tube) and store under foil at
4 degrees C.
Frozen Stocks (bacterial)
300 µl of overnight culture + 1.2 ml 80% glycerol (autoclaved).
Store @ -80°C
Freezing Media (mammalian cells)
Resuspend cell pellet in 1-1.5 ml of FBS, 10% DMSO. Freeze at @ -80°C
Extraction Buffer (MTT assay; for T cell Proliferation)
5 ml H2O
5 ml DMF (dimethlyformamide)
2 g SDS
Eosin
12 g Eosin Y
3 g Phloxine B (Sigma)
QS to 500 ml with 70% EtOH
EDTA (0.5 M)(disodium ethylene diamine tetraacetate)
186 g EDTA
800 ml H2O)
20 g NaOH
Stir until all EDTA goes into solution.
Adjust pH to 8.0 w/ NaOH
QS to 1 L
Elution Buffer (1X for Nickel Columns)
100 ml 500 ml
200 mM Imidazole 1.35 g 6.8 g
0.5 M NaCl 2.93 g 14.6 g
20 mM Tris HCL pH 8.0 2 ml 1 M stock 10 ml 2 M stock
pH to 7.9
Filter, store at 4°C
Extinction Coefficient
Σ280= (5600x #W) + (1400x #Y) + (200 x #F)
ELISA (example)
1-Coat plate with 0.1 µg target antigen in 50 µl PBS o/n @ 4°C. SEE ELISA TEMPLATE (triplicates).
Single Well Template.
NOTE: Peptides may need to be coated using Sodium Bicarbonate Buffer
2-Wash 3X with PBS 0.05% Tween 20. Typically this is done with an automatic plate washer or
hand-held washer. If not available you can use a squirt bottle to flush the wells. Crude but
effective.
After each wash step, tap plates (upside down) over a paper towel to remove an remaining wash
buffer.
3-Block with 200 µl/well SuperBlock (SB) (Pierce). 10% BSA dissolved in PBS is also okay but
SuperBlock works!!!
-Make all dilutions of antibodies from this step forward in SB
(or whatever your blocking agent is.
-Vortex all antibodies or reagents added to respective wells.
4-Wash 3X with PBS 0.05% Tween 20.
5-Add 100 µl of primary antibody. Depending on what this is it can be diluted 1:100-1:10,000.
Incubate at 37°C for 1 h.
6-Wash 5X with PBS 0.05% Tween 20.
7-Add 100 µl of secondary antibody. For this example assume it is alkaline phosphatase-
conjugated. Typically this can be diluted between 1:1000 and 1:5000. Incubate at 37°C for 1 h.
8-Wash 5X with PBS 0.05% Tween 20.
9-Develop using 1 Sigma Phosphatase tablet dissolved
in DEA. 1 tablet/5 ml DEA (See DEA protocol). 100 µl/well.
Develop between 30 min up to 3 h.
10-Read O.D. @ 405 nm.
E=mc2 Explained
F
Ficoll
This protocol written in the context of 'cleaning' up dead cells following a T cell stimulation with
irradiated/mitomycin C-treated APCs+Antigen.
1-Warm Ficoll to RT.
2-Warm the centrifuge to RT and set Acceleration and Brake to Zero.
3-Pipet 3 ml Ficoll into a 15 ml conical and overlay with 5 ml of media containing cells.
4-Centrifuge for 15 min.
5-Carefully pipet out the cells at the Ficoll-media interface.
6-Wash cells at least 3X with complete CTL.
Flow Cytometry
GENERAL FLOW CYTOMETRY PROTOCOL
Materials needed
FACS Buffer
PBS
.1 % BSA
.01% NaN3
Fetal Bovine Serum (FBS)
Primary antibody (biotinylated, fluorescently labeled, or unlabeled)
Secondary antibody (biotinylated or fluorescently labeled)
3rd stage (Streptavidin –fluorescent conjugate)
For multiple color staining fluorescent tags can be: GFP, FITC or Alexa 488 (FL1)
PE (FL2)
CyChrome or PerCP (FL3)
Procedure
- Wash cells 1X with at least 4 volumes of PBS
- Resuspend cells at 1 X 10_6 cells/ml in FACS buffer, put 1 ml/tube in Fisher 5 ml polystyrene stubs
- Block the cells in FACS buffer + 10% FCS for 15-30 minutes on ice
- Add 4 ml of FACs buffer to each tube, centrifuge 5 min at 2000 rpm
- Discard supernatant
- Vortex cells (there is approximately 100µl of liquid in each tube)
- Add appropriate amount of primary antibody to each tube (100-200 µl final volume in the tube)
most Abs work around 10 µg/ml
- Vortex tubes, incubate on ice for 30 minutes COVERED
- Add Secondary antibody at appropriate dilution to the tubes (most Abs work around 10 µg/ml)
- Add 4 ml FACs buffer to each tube, centrifuge 5 min @ 2000 rpm, repeat wash 2 more times
- Discard supernatant and vortex tubes
- If necessary, add Streptavidin at 1:1000 dilution (100-200 µl final volume in the tubes)
- Incubate on ice 20 min COVERED
- Add 4 ml FACs buffer to each tube, centrifuge 5 min @ 2000 rpm, repeat wash 2 more times
- Discard supernatant and vortex tubes
- Add 400 µl FACS buffer to each tube and analyze on flow cytometer immediately or fix in
paraformaldehyde (200 µl FACS + 200 µl 2% paraformaldeyde in each tube) and store under foil at
4 degrees C.
Frozen Stocks (bacterial)
300 µl of overnight culture + 1.2 ml 80% glycerol (autoclaved).
Store @ -80°C
Freezing Media (mammalian cells)
Resuspend cell pellet in 1-1.5 ml of FBS, 10% DMSO. Freeze at @ -80°C
| Protocols E-F |
