公司动态
Mouse ELISPOT-Assay
点击次数：443 发布时间：2012/6/20 16:47:09
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 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）
