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To check frequency of cytokine (IFN-γ '' IL-5'' IL-2'' IL-10'' IL-4) positive cells following in vitro Ag-specific activation.

 1. Set up 96-well plate map

 2. Pre-coating of ELISPOT 96-well plates with primary antibodies:

 

- new "white“ plates from Whatman/Polyfiltronics'' or white plastic nitrocellulose plates from Millipore

- 100µl of sterile PBS with     - anti-mIFN-γ (R46A2 hybridoma or Pharmingen)'' final 4µg/ml

                                              - anti-mIL-2 (Pharmingen)'' final 2µg/ml

                                               - anti-mIL-4 (11B11-hybridoma or Pharmingen)'' final 4µg/ml

- anti-mIL-5 (TRFK-5-hybridoma or Pharmingen)'' final 5µg/ml

                                                - anti-mIL-10 (not tested yet – Pharmingen)'' final ? µg/ml

- incubate o/n'' 4° humidified chamber (Tupper'' with soaked paper towels) C''

 

3. Before getting the mice preparation of:

 

Collection of blood/serum samples

Bleeding of mice by cardiac puncture: prepare Halothane glass cylinder'' cotton'' syringes'' needles'' and

eppendorf vials labeled.

 

cHL-1 medium;

calculation of volume:   a) proliferation assay = # organs x # antigens x # replicates x 0.1ml

b) cytokine supernat. = # organs x # antigens x # replicates x 0.5ml

c) ELISPOT = # cytokines x # organs x # antigens x # replicates x 0.1ml

 

x # of cell suspensions   d) spleen; 107/ml (final cell concentration = 106/well)

e) LN; 5x106/ml

 

Add L-glutamin'' and Pen/Strep'' sterile filter. (when not testing ex vivo peptide response'' one can also successfully use RPMI or DMEM'' 10% FCS; with other antigens!)

1) continue preparing ELISPOT plates

   - wash plates 3x with sterile PBS (200µl)

   - block 1hr with sterile PBS with „highgrade“ (cell culture'' BSA fraction V'' Sigma ) 1% BSA (200µl)

   - wash 3x with sterile PBS (200µl)

2) Plate out the antigens in fresh cHL-1 in ½ of the final volume (at 2x concentration):

a) medium (cHL-1)

b) Ovalbumin: OVA final 13.5µg/ml; „optimal“ for OVA T cell clones = 2.7µg/ml

c) Concanavalin A (final 2µg/ml) = from stock (100µg/ml stock)

 

in this case with Balb/c for relevant (I-A ) and irrelevant (I-A ) spleen APC or DC

in respective replicates of ELISPOT plates

 

And'' into 96-well (triplicates) for proliferation assay (same cell number as ELISPOT)

and'' into 2x24-well plates (pipette 500µl of 2x Ag solution + 500µl of 107/ml cell solution) or 2x96-well

plates for generation of cytokine supernatants (48hrs). Store at 4° until 1hr before the cells will beC

added'' then pre-warm in incubator at 37°C.

 

Warm up cHL-1 medium to 37°C.

 

Prepare for organ preparation: keep instruments in 75% EtOH (or flame); prepare petri dishes (# of

organs x 10ml DMEM); and 50ml tube top cell filter'' syringes to mash organs.

 

Get mice; do blood collection first'' then by neck dislocation kill groups of 3-8 (depending on organ

preparation time)'' mash organs with stamp of syringe'' pour through cell filter into 50ml tube.

 

Count cells (or estimate 80x106 per spleen with „normal“ size): set up fluorescence microscope (sign

on/off etc. needs at least 20min to warm up); get acridine orange / ethidium bromide dye solution'' mix 1:2

(to 1:10) of cells with dye (put dye on parafilm or microtiter plate)'' count green cells (living=acridin orange

cells) and red (ethidium bromide = dead cells)'' Put antigen loaded 96- and 24-well plates into 37° incubator. C

 

Spin cells 10min'' RT'' 1200rpm; resuspend with proper volume of cHL-1 to get final cell concentration of

spleen cells = 107/ml and LN = 5x106/ml.

Get antigen loaded plates and pipette (use tips with wide opening!!) the second ½ of volume to microtiter

or 24-well plates; do the magic shake! (= tap the plates slightly at both sides in order to distribute cells

evenly on the bottom of the wells) and

incubate at 37° 7% CO2.:     - IFN-γ '' IL-2'' for 24hrs

              - IL-4 and -5 for 48hrs (mostly from afternoon to the second daymorning = 40hrs; be careful not to disturb cells while incubating)

 

 

1) Continue after 24hrs with IFN-γ plates'' later or next morning IL-5 plates (do not bang the ELISPOT

   plates'' but shake out carefully):
- wash 3x PBS

- wash 4xPBS/Tween (0.05% = 500µl in 1L)'' let sit in the last wash for at least 5min

- remove washing solution

- add secondary antibody in sterile 100µl PBS/Tween/1% BSA

for mIL-2:       Biotin-labeled   = Biotin-rat anti-mouse IL-2'' Pharmingen'' 0.5mg/ml'' final  2µg/ml

for mIL-4:       Biotin-labeled   = Biotin-rat anti-mouse IL-4'' Pharmingen'' 0.5mg/ml'' final  4µg/ml   

for mIL-5:       indirect         = TRFK-4 (hybridoma'' or Pharmingen)'' final 4µg/ml

for mIFN-γ :     HRP-labeled      = XMG1.2-HRP (Pharmingen) final (= 1µg/ml)

- incubate in humidified box (Tupper) o/n at 4°C

 

anti mIFN-γ „direct“ (= XMG1.2 conjugated with HRP)

- wash IFN-γ (XMG1.2-HRP) plates/wells 3x PBS'' leave PBS

- AEC solution:  take 24ml AEC buffer'' pH 5.0 (storage)

              add 0.8ml AEC (= ImmunoPure AEC'' 3-amino-9-ethylcarbazole'' Pierce #34004'' 100mg in

              10ml dimethylformamide (DMF)'' chemical cabin'' RT'' wear gloves!)

              sterile filter with 150ml filter units (.45µm) before adding to plate: add 12µl of H2O2 (stock 30%'' Fisher # H325-100'' stored at 4°C)

- shake out plate

- add 200µl/well of AEC solution

- after 15 (IFN-γ) or 45-60min shake out plate

- wash with tap water 3x'' let dry (hood)

 

anti mIL-5 (unlabeled)

- wash IL-5 (unlabelled TRFK-4) plates gently 3x PBS/TWEEN (with squirt bottle)

- add mouse anti-rat IgG2a-HRP (ZYMED'' South San Francisco'' FAX (415) 871-4499; # 03-9620'' 1ml''

 HRP-mouse mAb anti-rat IgG2a)'' coupled mAb (frige 4° at sink) diluted 1:300 in PBS/TWEEN-BSA 1%C

 (40µl in 12ml)

- incubate at least 2hrs'' RT

- wash 3x PBS'' leave PBS

- then add substrate.... see above

 

anti mIL-2'' -4'' -10-Biotin

- wash plates gently 3x PBS/TWEEN'' let sit in wash #4

- add Streptavidin-HRP (Dako; frige 4° at sink)'' 1:2000'' in PBS/Tween/1%BSA'' 200µlC

- incubate at least 2hrs'' RT

- wash 3x PBS'' leave PBS

- then add substrate.... see above

 

Development

a) XMG-HRP: is turning dark'' but lightens with drying

b) XMG-Biotin: does not light up with drying!!

(in general: leave substrate at least 15 but at max. 60min)