在这里,我们展示如何准备从大鼠脾细胞培养上清TCGF。在此过程中,我们收获脾脏从幼稚刘易斯安乐死大鼠胸腺和血液采集。我们准备从脾单细胞悬液,析渗透压冲击的红血细胞,而种子在培养基中的脾。与非选择性地激活所有大鼠T淋巴细胞,诱导细胞因子的产生,有丝分裂原刀豆蛋白A的细胞受到刺激。文化supernantant收集48小时后andexcess伴刀豆球蛋白A是必然的α甲基mannoside,以防止它激活T细胞,TCGF将增加线路。 TCGF是无菌过滤,分装,储存于-20 ° C
Add 5 ml of 100x solution to 500 ml PBS to prepare PBS-PS
Cell strainer, 70 um diameter
Tool
Fisher
08-771-2
Ammonium Chloride (NH4Cl)
Reagent
Sigma
A0171
Prepare a 0.15 M solution in sterile distilled water, keep cold
RPMI 1640
Reagent
Gibco / Invitrogen
21870-092
Fetal Bovine Serum (FCS, FBS)
Reagent
Gibco / Invitrogen
16140-071
Heat-inactivated
Penicillin / Streptomycin / L-Glutamine
Reagent
Cambrex / Biowhittaker
17-718R
PSG
RPMI vitamins, 100x
Reagent
Sigma
R7256
Sodium pyruvate, 100x
Reagent
Sigma
S8636
Non-essential amino acids, 100x
Reagent
Sigma
Beta-mercaptoethanol
Reagent
Sigma
M7522
alpha methyl mannoside
Reagent
Sigma
M6882
Concanavalin A
Reagent
Sigma
M6882
To prepare complete culture medium add the following to a 500 ml bottle of RPMI 1640 and sterile-filter: 10% FCS; 1 bottle of PSG; 5 ml RPMI vitamins; 5 ml sodium pyruvate; 5 ml non-essential amino acids; 50 uM beta-mercaptoethanol; 2 ug/ml Concanavalin A.