Mammalian cell

Cell Seeding

์‹คํ—˜์— ํ•„์š”ํ•œ ์ •ํ™•ํ•œ ์„ธํฌ ์ˆ˜๋ฅผ ๊ณ„์‚ฐํ•˜์—ฌ ์ ํ•ฉํ•œ well dish์— ์„ธํฌ๋ฅผ ๋ถ„๋ฐฐํ•˜์—ฌ ์„ธํฌ ๋ฐฐ์–‘ํ•œ๋‹ค.

์ค€๋น„๋ฌผ

Complete media:

PBS:

Trypsin-EDTA (T/E)

์‹คํ—˜ ๊ณผ์ •

  1. Complete media, PBS, T/E๋ฅผ ๊บผ๋‚ด์„œ 37โ„ƒ water bath์— 10~15๋ถ„ ๋„ฃ์–ด์„œ ๋ฏธ๋ฆฌ ๋ฐ์›Œ ๋‘”๋‹ค.

  2. Suction์œผ๋กœ cell dish์˜ media๋ฅผ ์ œ๊ฑฐํ•œ๋‹ค.

  3. PBS๋กœ wash ํ›„, suction์œผ๋กœ ์ œ๊ฑฐ โ‡’ ์ฃฝ์€ ์„ธํฌ, ์ด๋ฌผ์งˆ, ๋‚จ์€ media

  4. Trypsin EDTA๋ฅผ ๋„ฃ๊ณ  ๋ถ€๋“œ๋Ÿฝ๊ฒŒ ํผ๋œจ๋ฆฌ๊ณ , Incubator 1~2๋ถ„ โ‡’ adhesion ๋‹จ๋ฐฑ์งˆ์„ ๋–ผ์–ด๋‚ด ์คŒ

  5. 15ml ํŠœ๋ธŒ(tube)์— 9ml media + 1ml cell containing trypsin EDTA solution์„ ์ตœ๋Œ€ํ•œ ๋ชจ์•„ ๋„ฃ๋Š”๋‹ค

  6. ์›์‹ฌ ๋ถ„๋ฆฌ(Centrifuge) = 1000 rpm์œผ๋กœ 3๋ถ„ ๋Œ๋ ค์„œ cell pellet์„ ๋งŒ๋“ ๋‹ค.

  7. ์ƒ์ธต์•ก์„ ์ œ๊ฑฐํ•˜๊ณ , tube์— 5ml media ์ฒจ๊ฐ€ํ•ด์„œ ์„ธํฌ๋ฅผ ํ’€์–ด์ค€๋‹ค.

  8. Cell counting โ‡’ 10 ฮผl cell media + 10 ฮผl 0.4% trypan blue stain(์—ผ์ƒ‰์•ฝ)

  9. ๊ฐ well๋งˆ๋‹ค 3ml media ์ฒจ๊ฐ€ โ‡’ 6well ๊ธฐ์ค€, ๊ฐ well๋งˆ๋‹ค = media 3ml (๊ณ ์ •๊ฐ’) + counting cell (ฮผl). cell counting ๊ณ„์‚ฐ ๊ฒฐ๊ณผ์— ๋งž๋Š” ์–‘์„ ํ”ผํŽซ(pipette)์œผ๋กœ ๋”ฐ์„œ dish์— ๋„ฃ๋Š”๋‹ค.

  10. ์„ธํฌ๊ฐ€ dish์— ์ ์ ˆํ•˜๊ฒŒ ์žˆ๋Š”์ง€ ํ˜„๋ฏธ๊ฒฝ์œผ๋กœ ๊ด€์ฐฐ

  11. Incubator์— ๋„ฃ๊ณ  ๋ฐฐ์–‘ํ•œ๋‹ค.

Cell line๋งˆ๋‹ค ๋ฐ”๋‹ฅ์—์„œ ๋–จ์–ด๋œจ๋ฆฌ๊ธฐ ์œ„ํ•ด ์ฒ˜๋ฆฌํ•ด์•ผ ํ•˜๋Š” trypsin EDTA ์–‘์ด ๋‹ค๋ฅด๋‹ค. ์˜ˆ๋ฅผ ๋“ค์–ด, HEK293T cell line์€ ์ž˜ ๋–จ์–ด์ง€๋Š” ํŽธ์ด๋ฉฐ, 100-mm dish ๊ธฐ์ค€, Trypsin EDTA 500uL์ด๋ฉด ์ถฉ๋ถ„ํ•˜๋‹ค.

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