Conversion of surface area and volume for various cell culture formats

Materials/Reagents:
?   Sulfo-SMCC (succinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate, Thermo scientific) MW=436.37 Da
?   Hydroxylamine-HCl (Pierce)
?   SATA (N-succinimidyl S-Acetylthioacetate, Pierce) MW=231.23
?   Reaction Buffer EDTA-free pH7.2-7.5
   100mM Sodium Phosphate
   150mM NaCl
?   Reaction buffer + EDTA same pH as other Reaction buffer (7.2-7.5)
   100mM Sodium Phosphate
   150mM NaCl
   10mM EDTA
?   Conjugation buffer
50% Reaction buffer +10mM EDTA
50% Reaction buffer NO EDTA
?   De-Acetylation solution (make in Reaction buffer, EDTA-free), pH to same as Reaction buffer
   0.5M Hydroxylamine (1.74g in 50mL)
   25mM EDTA (0.475g tetra or 0.365g disodium salt in 50mL)
?   IgG of choice
?   IL-2 or other appropriate cytokine
?   1mg of IL-2 is equivalent to 16.9 million Units and at 15,517 Da is equivalent to 64.4nMoles IL-2.

1.   Dialyze 2mg of antibody into reaction buffer (NO EDTA) extensively and concentrate down to 9mg/mL final (222uL for 2mg).
2.   Dissolve 10mg SATA in 625uL DMSO.
3.   Add 3uL SATA solution to 222uL antibody solution.
4.   Incubate for 30 minutes at RT.
5.   Dialyze away excess SATA using reaction buffer (NO EDTA) and bring volume to 1mL.
6.   Make up fresh de-acetylation solution and add 100uL to 1mL sample to deacetylate SATA.
7.   Incubate for 2 hours at RT.
8.   During incubation of SATA antibody, perform steps 9-13
9.   Resuspend 1mg IL-2 in 1mL conjugation buffer.
10.   Resuspend Sulfo-SMCC to 4.8mg/mL final.  (ie 10.42 mL for 50mg)
11.   Add 29uL of sulfo-SMCC mixture to 1mg IL-2 in conjugation buffer.
12.   Incubate reaction for 30 minutes at RT.
13.   Concentrate or dialyze away excess crosslinker (MW 436.37, IL-2=15,517) with conjugation buffer extensively.
14.   Dialyze SATA modified antibody into Reaction buffer (10mM EDTA) extensively.
15.   Combine SATA-Antibody and Sulfo-SMCC-IL-2.
16.   Incubate at overnight at RT.
17.   Dialyze to PBS extensively using dialysis tubing of at least 30kDa to remove excess IL-2, 50-100kDa tubing works best.

Protocol by Eric Mortensen

1.   Isolate LN and spleen on bench placing them into wells of a 6-well plate w/strainer and 5 mL of media
a.   For one animal spleen and LN can be mixed in one well, but for more animals use separate wells for LN and spleen
2.   In the hood, mash spleen and LN with the plunger of a 1mL syringe
3.   Transfer to a 50mL conical vial through filter
4.   Fill tube with 50mL of PBS, washing original well also
5.   Centrifuge 1500rpm 5min
6.   Pour off PBS and resuspend in 1-2 mL ACK per spleen
7.   Incubate for 3 minutes
8.   Stop reaction by filling tube with PBS
9.   Take a small sample for counting (10uL) and for 2 samples for FACS (200uL)
10.   Centrifuge 1500rpm 5min
11.   Pour off supernatant and re-suspend cells in PBS to a concentration of 2x107/mL (up to 5x107/mL)
12.   Cover tube in foil
13.   Add stock CFSE (10mM) into the cell suspension for a final concentration of 10M for in vivo adoptive transfer experiments
14.   Mix well keeping tube covered in foil
15.   Incubate cells with CFSE for 30 min at 37C
16.   Stain cells during incubation
a.   Add 0.5uL of 1B2-FITC (# 483) to one tube leave other for unstained control
b.   Incubate 15-20 minutes at 4C in dark
c.   Wash with 2mL FACs buffer and spin
d.   Decant and resuspend in 200uL FACS buffer
17.   Quench CFSE by adding an equal volume of FCS into cell suspension mix well and incubate RT for one minute
18.   Add media (RPMI) to fill tube and filter into a new tube
19.   Take small sample (10uL) for counting and centrifuge 1500rpm 5min
20.   Pipette off all media and resuspend in an appropriate volume of PBS to have 1-2x106 Transgenic Cells injected in 0.1mL (need to use the staining to calculate!)
21.   Inject 0.1uL per mouse IV
22.   Can check CFSE staining efficiency by FACS on left over cells

Electroporation of Mammalian cells

Method 1
1.   Mix 30ug DNA with 7x106 cells in 300uL in 4mm gap cuvette in RPMI with 10%FBS and 1.25% DMSO.
2.   Set conditions to 960uF and 250-290 volts.
3.   Pulse cells and rescue in 10mL media with 1.25% DMSO, incubate 24 hours at 37.
4.   Change media after 24 hours into normal media, incubate 24 hours.
5.   Put cells under selection, for transient, take samples every 24 hours.

Method 2
1.   Mix 50ug DNA with around 1x107 cells in 4mm gap cuvette.
2.   Set pulse controller to 200 ohms, capacitance at 950uF at 300 volts.
3.   Pulse cells, set on ice for 10 minutes.
4.   Resuspend in 10mL of complete media with 1% DMSO, incubate at 37.
5.   Change media after 24 hours to media without DMSO.
6.   After 48 hours (post electroporation), put cells under selection.

Method 3
1.   4x106 cells in 800uL of serum-free RPMI in 4mm gap cuvette.
2.   Settings at 500uF and 330V, the rest is same as above.

Method 4
1.   Mix 30ug DNA with 1x107 cells in 400uL of PBS with 6mM dextrose and 1.25% DMSO.
2.   Set conditions to 1050uF, infinite resistance, voltage between 200 and 350 volts.
3.   Pulse cells and recover as above.

IFN- ELISpot assay by Olga Brown

Reagents:
Sterile 96-well PVDF plates
70% ethanol in sterile water
Sterile PBS
Non-sterile PBS
Coating Ab (BD cat 554430): purified anti-mouse IFN- (NA/LE sterile) in PBS, 1:200-1:400 dilution
Blocking buffer: 1% BSA in PBS, sterile filtered
ELISpot medium: RPMI 10% FBS, HEPES, P/S, NEAA, L-Glutamine, sterile filtered
Wash buffer:  PBS with 0.05% Tween-20
Dilution buffer: 1% BSA in PBS with 0.05% Tween-20, sterile filtered
Detection Ab (BD cat 554410): biotinylated anti-mouse IFN- in dilution buffer, 1:250 dilution
Streptavidin-HRP conjugate (BD cat 554066): SAv-HRP in dilution buffer, 1:1000 dilution
AEC substrate (BD cat 551951): 20 l of AEC chromagen per  1 ml of AEC substrate diluent; add chromagen to diluent, not vice versa. 

TAKE GREAT CARE NOT TO PUNCTURE THE MEMBRANE ON THE BOTTOM OF THE ELISPOT PLATE.
ADD REAGENTS TO WELLS CAREFULLY AND WITHOUT FORCE.

Coating ? in the hood!

1.   Prepare coating Ab and 70% ethanol solution.
2.   Add 100 l/well of 70% ethanol in sterile water. Incubate plates for 2-10 min at RT.
3.   Discard alcohol and wash wells 4x with 130 l/well of sterile PBS.
4.   Add 100 l/well of coating Ab.
5.   Store plates at 40C overnight and no more than 3 days.

Blocking and cell activation ? in the hood!

1.   Discard coating Ab and wash wells 1x with sterile PBS.
2.   Add 150 ul/well of sterile blocking buffer. Incubate plates for 2 hrs at RT.
3.   Discard blocking buffer and wash wells 4x with 130 ul/well of sterile PBS.
4.   Prepare responder cell suspensions at 4 X 106 cells/ml. Carefully and slowly add 100 ul/well to the center of ELISpot plate microwells.
5.   Prepare antigen or APCs, diluted in ELISpot medium. When using tumor cells as a source of antigen, use at 1:10 tumor : lymphocyte ratio. Add 100 ul/well of 2x antigen solution or 4 x 105 cells/ml tumor cells. As controls, add 100 ul/well ELISpot medium or 100 ul/well  of 4 ug/ml ConA in ELISpot medium. Add cells or antigen solution very carefully to the center of the ELISpot plate microwells in the direction opposite to the addition of lymphocytes.
6.   Incubate plates at 370C 5% CO2 for 24-48 hrs. DO NOT STACK PLATES IN THE INCUBATOR!  DO NOT MOVE OR DISTURB PLATES DURING INCUBATION!

 
Detection ? in the hood/on the bench

1.   Discard cells and blot plates on a paper towel. NEVER PLACE THE BOTTOM OF AN ELISPOT PLATE ON A PAPER TOWEL ? IT WILL ABSORB THE CONTENTS OF THE WELLS!
2.   Add 150 ul/well of wash buffer. Incubate for 10 min at 40C.
3.   Prepare detection Ab ? KEEP DILUTION BUFFER STERILE!
4.   Wash wells 3x with 150 ul/well of wash buffer.
5.   Add 100 ul/well of detection Ab. Incubate plates for 2 hrs at RT.
6.   Discard detection Ab and wash wells 4x with 150 ul/well of wash buffer.
7.   Add 100 ul/well of Streptavidin-HRP in dilution buffer ? KEEP DILUTION BUFFER STERILE! Incubate plates for 2 hrs at RT.
8.   Discard streptavidin-HRP and wash wells 3x with 150 l/well of wash buffer.
9.   Wash wells 3x with 150 ul/well of PBS.
10.   Blot plate on a paper towel.
11.   Add 50 l/well of AEC substrate.
12.   Incubate for 5 min at RT (if color develops fast, only 3 min)
13.   Wash plate with MiliQ water from the tube, not pipette, 6-7 times. Remove plastic bottom of the plate and wash with MiliQ water from the tube 4-5 times.
14.   Blot plate (top and bottom) on a paper towel.
15.   Place plate to dry on the mesh part of the hood without lights or covered with a paper towel.
16.   When plate is completely dry (membranes and plastic between wells), read plate immediately or wrap in foil and store at -200C.

PEI Transfection of CHO cells, 6-well format (for scaling up, see scale up document for formulas)

1.   Seed CHO cells at 3x105 cells per 3.5cm dish (or 1 well of a 6-well plate) 24 hours before transfection.
2.   Change media on day of transfection to 0.9mL serum-free glutamine-free media.
3.   Mix 8uL PEI(1mg/mL) with 1ug DNA (can use up to 3ug DNA and 24uL PEI for transients before large scale cell-death) in 100uL of optimem, mix and incubate 10 min RT.
4.   Add to cells dropwise.  Incubate 3-4 hours at 37.
5.   Change to growth media and incubate 24-96 hours.  Take samples periodically.

Some of the text did not show up, here is the actual document

Alternative Cell Lysis Buffers

Notes: A single cell-lysis buffer may not work for every application.  Considerations must be made such as what types of cells are used, the cellular location of the protein, the strength of the protein:protein interactions and solubility issues.  Protease and phosphatase inhibitors have been omitted from these recipes so be sure to add them back before use.

Lysis buffer 1
•   50mM Tris-HCl pH 8.8
•   100mM NaCl
•   5mM MgCl2
•   0.5% NP40 or IGEPAL
•   1mM EDTA

Lysis buffer 2
•   150mM NaCl
•   50mM TrisHCl
•   0.1mM CaCl2
•   1% Triton X-100

Western Blot Protocol (Standard, using Bio-RAD system)
Protocol is similar to Cell-Signaling Tech protocol

Notes: Either BSA or non-fat dry milk can be used to block blots.  Primary antibody concentrations that are too high may not be completely washed away in first three washes and could bind secondary antibody and be subsequently washed away, lowering signal.  Directly labeled HRP or biotinylated antibodies work best (with SA:HRP secondaries) and give lower backgrounds than anti-IgG secondaries, which usually give higher backgrounds.  Two quick washes before the three 5 minute washes can be used to lower membrane background after secondary or primary if desired.  Super high membrane background (membrane outline background) can be eliminated by washing membrane in wash buffer containing a small amount of sodium azide (0.03% final) for initial 1 or two washes after secondary incubation.  Be sure to not use sodium azide wash buffer in final wash, it will greatly inhibit HRP activity in the presence of substrate.

Buffers/Reagents
•   Transfer Buffer: 25mM Tris Base, 0.2M glycine, 20% Methanol (pH 8.5)
•   10x TBS
•   Blocking buffer: 1X TBS, 0.1% Tween-20, 5% non-fat dry milk or BSA
•   Wash Buffer: 1X TBS, 0.1% Tween-20
•   Antibody Incubation Buffer: 1X TBS, 0.1% Tween-20, 1-5% non-fat dry milk or BSA (user determined for background vs signal)
•   ECL reagents (If using HRP), see protocols for homemade ECL

1.   After running gel, equilibrate all transfer materials in transfer buffer for 15-30 minutes.
2.   Assemble transfer apparatus. For Bio-RAD wet transfer, assemble in following order: clear side, sponge, filter paper, membrane, gel, filter paper, sponge, black side. Be sure to eliminate all bubbles and keep sandwich wet while assembling.
3.   Put sandwich into wet-transfer apparatus by matching black side of sandwich to black side inside transfer box and transfer at 100V for anywhere between 30 minutes and two hours depending on protein size and type of membrane used.  Be sure to use ice-cold transfer buffer and an ice pack since transfer creates lots of heat. A stir bar in the bottom helps prevent hot zones.
4.   After transfer, wash membrane in 1X TBS at RT for 5 minutes.
5.   Incubate membrane in blocking buffer for 30 minutes to 1 hour at RT.
6.   You can wash the membrane after blocking or simply dump out blocking solution and add primary antibody solution.  Concentration of primary should be determined for each antibody that is used.
7.   Incubate overnight at 4 C with gentle agitation.
8.   Wash membrane 3 times with Wash buffer, for 5 minutes each wash.
9.   Incubate membrane with secondary antibody (if appropriate), 1 hour RT.
10.   Wash 3 times with Wash buffer, 5 minutes each wash.
11.   Incubate membrane with ECL reagents of choice.
12.   Expose x-ray film with membrane, times vary greatly from 1s to overnight depending on strength of signal.  Try to get multiple exposure times.
13.   Label film.
14.   Membrane can be stripped, re-probed or stored at 4 C.

Slight additions made.