細(xì)胞ELISA實驗 Cellular ELISA Protocol.doc

Cellular ELISA ProtocolAuthor:Nanci DonackiSource:Contributed by Nanci DonackiDate Added:Tue May 14 2002Date Modified:Wed Apr 28 2004Formalin Fixed Cell Plates1. Trypsinize confluent flasks2. Pool and count cells3. Centrifuge at 1500 rpm for 10 minutes4. Resuspend to the appropriate concentration in complete medium4 x 105 cells/ml for epithelial cells2 x 105 cells/ml for fibroblast cells5. Add 100 ml/cell to 96 well culture plates.6. Incubate overnight at 37oC.7. Wash plates twice with PBS8. Add 125 ml/well 10% Buffered Formalin9. Fix for 15 minutes at room temperature10. Wash three times with di-H2O.11. Blot dry.12. Store at 2-8oC.Reagents1. PBS:1% BSA2. PBS:2% BSA3. Carbonate Buffer1.59 gNa2CO32.93 gNaHCO3Dissolve in 900 ml di-H2O. Check pH and adjust to 9.6 necessary. Qs. to 1 liter.4. 10X Substrate Buffer, pH 6.036.6 gCitric Acid, monohydrate113.5 gPotassium dibasic phosphateDissolve in 900 ml di-H2O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter.5. 0.3% H2O2Dilute 30% stock Peroxide 1:100 in di-H2O.6. OPD Stock, 4.0%4 g OPD in 100 ml di-H2O. Aliquot and store at -20oC. Protect from light.7. 4.5N H2SO412.0 mlConcentrated Sulfuric Acid88.0 mldi-H2OProcedure1. Wash ELISA plates once with di-H2O.2. Add 250 ml/well PBS:2% BSA.3. Incubate 1 hour at 37oC.4. Wash 3 times with di-H2O.5. Add 50 ml/well supe, ascites, or controls diluted in PBS:1%BSA.6. Incubate for 2 hr at 37oC.7. Wash 5 times with di-H2O.8. Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:1% BSA.9. Incubate for 1 hr at 37oC.10. Wash 5 times with di-H2O. Wash once with carbonate buffer.11. Add 50 ml/well working substrate solution0.5 ml4.0% OPD5 ml30% H2O21.0 ml10X Substrate buffer8.5 mldi-H2O.12. Incubate for 20 minutes at room temperature.13. Add 25 ml/well 4.5N Sulfuric Acid14. Read A490Notes1. Test all supernatants at 1:5 dilution.2. Test ascites at 1:100細(xì)胞ELISA實驗一、福爾馬林固定細(xì)胞1、用胰蛋白酶消化細(xì)胞培養(yǎng)瓶中的細(xì)胞；2、收集細(xì)胞并計數(shù)；3、離心，1500rpm，10min；4、用完全培養(yǎng)基重懸細(xì)胞于適當(dāng)?shù)臐舛龋掀ぜ?xì)胞4105個細(xì)胞/ml，纖維細(xì)胞2105個細(xì)胞/ml；5、滴加到96孔培養(yǎng)板中，100ul/孔；6、37C環(huán)境孵育過夜；7、用PBS洗板兩次；8、每孔加125ul 10福爾馬林緩沖液；9、于室溫環(huán)境下固定15min；10、用雙蒸水洗滌三次；11、晾干；12、貯存于2-8 C.二、細(xì)胞ELISA實驗試劑：1、PBS：1 BSA2、PBS：2BSA3、碳酸鹽緩沖液：1.59g Na2CO32.93g NaHCO3溶解于900ml雙蒸水中，檢測并調(diào)整pH至9.6，定容至1L；4、10底物液，pH6.036.6g一水合檸檬酸113.5g 磷酸氫二鉀溶解于900mL雙蒸水中，調(diào)pH至6.0，定容至1L；5、0.3H2O2：用雙蒸水按1：100稀釋30的雙氧水6、OPD貯存液，4.0：4g OPD溶解于100ml雙蒸水，分裝后保存到-20 C,注意避光。7、4.5N H2SO412.0mL濃硫酸88.0ml雙蒸水混合三、實驗步驟：1、用雙蒸水洗滌ELISA板兩次；2、每孔加250ul含2BSA的PBS；3、37C孵育1h；4、雙蒸水洗板三次；5、每孔加50ul腹水，上清，或用含1BSA的PBS稀釋后的稀釋品；6、37C孵育2h；7、雙蒸水洗板五次；8、每孔加50ul用含1BSA的PBS稀釋的HRP酶標(biāo)抗鼠IgG；9、37C孵育1h；10、用雙蒸水洗板5次。用碳酸鹽緩沖液洗兩次；11、加工作濃度底物液50ul/孔，配方如下