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Biology

准备从大鼠脾细胞T细胞生长因子

doi: 10.3791/402 Published: October 31, 2007

Summary

我们描述的T细胞生长因子在体外扩增抗原特异性大鼠T淋巴细胞线的准备。

Abstract

抗原特异性T细胞株或文化克隆的维修需要轮抗原诱导激活细胞扩张 1,2阶段分开。白细胞介素2除了在扩张阶段的文化传媒,是必要措施,防止细胞死亡,并足以维持短期的T细胞系,但已被证明能增加Th1细胞极化3。 T细胞生长因子(TCGF),其中包含混合的细胞因子白细胞介素2的更换是更有效地保持长期的T细胞系在体外培养3比白细胞介素2。此外,TCGF可以很容易地在实验室中大量的准备和重组白细胞介素2比便宜得多。

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

Protocol

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  1. 以Lewis大鼠脾脏(160 - 200G之间的老鼠是最好的)。在培养皿菜含有PBS +抗生素(PBS - PS),使用细胞过滤器Dilacerate冰。放入50毫升管。填写用PBS - PS。
  2. 自旋为10分钟,在4 ° C至沉淀细胞。
  3. 清洗细胞两次。
  4. 悬浮颗粒在NH 4氯0.15米(5毫升每脾)。混合3分钟就冰吸管轻轻地,不断溶解红细胞。填充介质管。
  5. 自旋为10分钟,在4 ° C至沉淀细胞。
  6. 清洗细胞两次。
  7. 细胞计数。大鼠脾给出了200-250万个单元格。
  8. 种子细胞完全培养基,在每毫升200万:50毫升每75厘米2烧瓶。
  9. 让我们成长为48小时,在孵化器。
  10. 旋在4 ° C 15分钟沉淀细胞。
  11. 收集上清液;丢弃的细胞。
  12. 添加15毫克/毫升甲基mannoside去上清。调匀。
  13. 过滤器(0.2毫米)
  14. 分装并储存于-20 ° C 10天,如果必要,可以保持在4 ° C。

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Discussion

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我们准备从Lewis大鼠脾细胞TCGF,因为我们经常从天真这株收获血清和thymi Lewis大鼠T细胞系在体外刺激大鼠安乐死。这TCGF,可以被用来促进从其他的大鼠株T细胞系的生长和生存。 TCGF也可准备从其他菌株的大鼠。

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Materials

Name Type Company Catalog Number Comments
PBS, 1x, sterile Reagent GIBCO, by Life Technologies 14040-182
Penicillin / Streptomycin 100x Reagent Sigma-Aldrich P0781 Add 5 ml of 100x solution to 500 ml PBS to prepare PBS-PS
Cell strainer, 70 um diameter Tool Fisher Scientific 08-771-2
Ammonium Chloride (NH4Cl) Reagent Sigma-Aldrich A0171 Prepare a 0.15 M solution in sterile distilled water, keep cold
RPMI 1640 Reagent GIBCO, by Life Technologies 21870-092
Fetal Bovine Serum (FCS, FBS) Reagent GIBCO, by Life Technologies 16140-071 Heat-inactivated
Penicillin / Streptomycin / L-Glutamine Reagent Cambrex/BioWhittaker 17-718R PSG
RPMI vitamins, 100x Reagent Sigma-Aldrich R7256
Sodium pyruvate, 100x Reagent Sigma-Aldrich S8636
Non-essential amino acids, 100x Reagent Sigma-Aldrich
Beta-mercapt–thanol Reagent Sigma-Aldrich M7522
alpha methyl mannoside Reagent Sigma-Aldrich M6882
Concanavalin A Reagent Sigma-Aldrich 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-mercapt–thanol; 2 ug/ml Concanavalin A.

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References

  1. Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -M., Gola, M., Sabatier, J. -M., Bernard, D., Crest, M., Beraud, E. Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation. J. Immunol. 166, 936-944 (2001).
  2. Beeton, C., Wulff, H., Barbaria, J., Clot-Faybesse, O., Pennington, M., Bernard, D., Cahalan, M. D., Chandy, K. G., Beraud, E. Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. 98, 13942-13947 (2001).
  3. Mor, F., Cohen, I. R. Propagation of Lewis rat encephalitogenic T cell lines: T-cell-growth-factor is superior to recombinant IL-2. J. Neuroimmunol. 123, 76-82 (2002).
准备从大鼠脾细胞T细胞生长因子
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Cite this Article

Beeton, C., Chandy, K. G. Preparing T Cell Growth Factor from Rat Splenocytes. J. Vis. Exp. (10), e402, doi:10.3791/402 (2007).More

Beeton, C., Chandy, K. G. Preparing T Cell Growth Factor from Rat Splenocytes. J. Vis. Exp. (10), e402, doi:10.3791/402 (2007).

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