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物种
Mouse溶解方法
According to the instructions for each component
储存条件
IL-2
IL-6
TGF-β1
IL-21
·12 months from date of receipt, lyophilized powder stored at -20 to -80℃.
· 3 months, -20 to -80℃ under sterile conditions after reconstitution.
· 1 week, 2 to 8℃ under sterile conditions after reconstitution.
· Please avoid repeated freeze-thaw cycles.
Invivo anti-mouse CD3ε Recombinant mAb
NA/LE Syrian Hamster anti-mouse CD28 mAb
Invivo anti-mouse IFNγ Recombinant mAb
Invivo anti-mouse IL-4 Recombinant mAb
Invivo anti-mouse IL-2 Recombinant mAb
2 to 8 °C for 2 weeks under sterile conditions;
-20 °C for 3 months under sterile conditions;
-80 °C for 24 months under sterile conditions.
Please avoid repeated freeze-thaw cycles
Component
Reference dosage
10ml System（S Size）
100ml System（M Size）
1000ml System（L Size）
IL-2
5ng/ml
5μg
5μg
5μg
IL-6
100ng/mL
5μg
10μg
100μg
TGF-β1
30ng/ml
5μg
5μg
30μg
IL-21
50 ng/ml
5μg
5μg
50μg
Invivo anti-mouse CD3εRecombinant mAb
10µg/ml
100μg
1mg
10mg
NA/LE Syrian Hamster anti-mouse CD28 mAb
2μg/ml
20μg
200μg
2mg
Invivo anti-mouse IFNγRecombinant mAb
10μg/ml
100μg
1mg
10mg
Invivo anti-mouse IL-4 Recombinant mAb
10μg/ml
100μg
1mg
10mg
Invivo anti-mouse IL-2 Recombinant mAb
10μg/ml
100μg
1mg
10mg
1、细胞培养
用10 µg/ml anti-mouse CD3ε mAb和2 µg/ml anti-mouse CD28 mAb包被48孔细胞培养板过夜，第二天洗板封闭后加入CD4 Nanobeads（ S0K0003 ）分选后的CD4+ T细胞，培养基用RPMI-1640 加100 ng/ml IL-6, 50 ng/ml IL-21, 30 ng/mL TGF-β1, 10 μg/ml anti-IL-2, 10 μg/ml anti-IFN-γ,10 μg/ml anti-IL-4, 55 μM β-巯基乙醇及10 % FBS培养, 48 小时开始每 12 小时观测细胞浓度，如果细胞密度很大，则将细胞分到新的孔中，并加以上培养液刺激（不用添加 anti-mouse CD3ε和anti-mouse CD28 mAb刺激），培养5天后收集细胞细胞，将胞密度控制为10⁶/ml，用10ng/ml PMA,1μg/ml Ionomycin，10μg/ml Brefeldin A共处理5h。阴性对照组Th0细胞：用10 µg/ml anti-mouse CD3ε mAb和2 µg/ml anti-mouse CD28 mAb包被48孔细胞培养板过夜，第二天洗板封闭后加入分选后的CD4+ T细胞，维持培养基为RPMI-1640 加 10 % FBS, 55 μM β巯基乙醇，10μg/mL anti-mouse IFNγ，10μg/mL anti-mouse IL-4 培养，48 小时开始每 12 小时观测细胞浓度，如果细胞密度很大，则将细胞分到新的孔中，并加以上培养液（不用添加 anti-mouse CD3ε和anti-mouse CD28 mAb）培养5天。
2、流式检测
收集细胞后用流式检测抗体染色上机
S0B5654 starter APC Rat Anti-Mouse PD-1 Antibody
store in separate containers to reduce the number of freeze-thaw cycles.
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