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EMBRYONIC STEM CELLS PROTOCOL$ x9 k( L( I9 Y$ y/ b8 A- ]& L
Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作
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$ f+ s$ F z( h; h. J# r- [! _- jEMBRYONIC STEM CELLS PROTOCOL
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If you are embarking in growing ES cells, be prepared to refeed
6 }9 v3 x/ l& ` U" b0 d/ @7 k3 u2 mthem DAILY. All procedures should be carried out using sterile 9 m. |: h- C0 E7 F
techniques. The growth and maintenance media for ES cells is M15 MEM ( ^, J+ G1 d S- R" h
(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells:
5 ~9 C& _6 r0 P/ hgrowth, maintenance, passing, freezing and thawing is conducted in a * @) Q' x3 O( i6 d' G- e0 L, A
manner to protect and maintain the quality of the cells and keep them in
; ~9 [' p% v5 R. ~6 Oa pluripotential state. Serum quality is critical for successful growth / }+ F' M) j2 B/ K B' a& A
of ES cells and especially true for blastocysts. The quality of the 0 {9 ]- R) d: l- c9 x4 A$ i* |
feeders is very instrumental. Remember also that in passing, freezing,
& I$ d6 [* M+ mand electroporating ES cells; it is best that the cells are still at - k0 t8 X0 a, v2 J( J
exponential growth (80% confluence) for optimal results.
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THAWING (QUICK THAW)
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+ Q) R2 j, V, W) `$ W9 a8 ]1. Remove cells from the freezer and quickly thaw in a 37oC waterbath.
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( b+ R1 y( q1 ]; d, j- @: M2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - ( u! }( S, o# A$ ? S$ X' y
12 mls of M15 media to 1 ml of cell suspension.% C \0 p8 C& Q7 H* J; M& u& X% F
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3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.7 |) r" } w& a- r
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4. Aspirate off supernatant and resuspend cells into 6 mls of M15, $ M+ n4 ]) ? |1 L5 n9 K! U
and plate out cells in a 6-cm feeder plate.
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5. Refeed cells daily with fresh M15. Upon 80-85% confluence, cells
8 I8 W: N9 n1 B! Oneed to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME)
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PASSAGE OF ES CELLS7 I; B; @1 ~( z" y
\& n( B, p! _) x* B( n- bES cells typically should be passaged every 2-4 days (apart from colonies
( ]8 |" j0 X# z j8 b! C5 J9 Zunder selection). If passaging is neglected the cells will differentiate
+ I. x* \1 G5 H# m Y2 hand you will select for variants that might have lost totipotency. Cells
: v6 l1 L7 z& C$ D" |1 x- Kmust be fed when media begins to turn orange. Yellow media (acid pH) is ( N W! y6 \ v) A6 Y. \: J
very bad for ES cells and should be avoided at all costs. If you are
% [% l" X+ S2 U7 p- ^planning to passage and believe that the cells might turn yellow 3 J: N8 {1 {. w8 E
overnight feed last thing in the evening and again the next morning / ]0 f \0 @0 F- {8 _
before passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW.. p/ w9 e2 t$ z0 k* v
& p- |% N# S+ W0 t$ H1. Check cells under the microscope for 80-85% confluence.
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# R" |: `- D( X5 j2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT)
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3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
+ Q R# d% J9 l7 r ]& }- Rtrypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.: h# Y4 E3 Y3 v
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4. Incubate @ 37 oC for 15 minutes.
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; o7 ~ M) l! b. @5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x
0 ]' _& i# t" r6-cm dish or 4 - 5 mls to 1 x 10-cm dish.) I/ d: o( N! D6 ]9 M' {
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6. With a transfer pipette, pipet up and down several times to # R: D8 Y8 ~/ x" D+ J3 m( M2 z) [
separate the cells and break any colonies." t3 k$ w0 n7 w- F/ R
/ b. l ]- y! h* K7. Determine the number of feeder plates you need, depending upon # w( x4 t0 d- ]4 @
the passage you are doing. Add fresh media, M15 to the feeder plates (to
, \: _& \' o( }1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of
1 Z; a8 J9 E2 z! B# G4 Fmedia). Split ratios for ES cells can vary from 1:1 to 1:10. Do not
- v: H1 @6 W' n; a( Yexceed 1:10. : h; U4 k |$ N4 g2 c& {! k
The area relationships for the various dishes are as follows:0 }+ L4 I/ L" g q$ C$ z9 t
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Dish Media Trypsin Area (cm2) Diameter (actual)6 P: O8 b) N2 A+ M6 R* Q
(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm+ Q u0 Q, E( q* m+ V
(10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm, C+ d: Z p, b
(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm
8 X8 b$ c, c8 N2 t6 S4 V) p. I) i; f8 s6 G; M(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm
2 }. U- @# r4 o# }- q9 Y(10-cm) dish 12 mls 1.5 ml 60 8.7 cm+ b, l, h" k! V2 K0 r
(15-cm) dish 30 mls 2.5 ml 154 14 cm
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' h* e) R. L' {7 f H- c6 VSome typical passaging ratios:- q( d k, B* ?2 ~2 x) Q7 e
9 g; o$ a+ f( I2 P. s1:6 = 1 x 60 mm to 2 x 90 mm 2 @, j; q- {$ @1 m3 C1 @
1:6 = 1 x 30 mm to 1 x 90 mm( L+ c' ~% A" e. [
1:4 = 1 x 30 mm to 2 x 60 mm 3 b; ~2 b: X' c/ ^, g, z
1:5 = 1 x 24 well to 1 x 30 mm (6-well plate) 9 }; e+ f. E3 D* j* X- p: c
1:6 = 1 x 96 well to 1 x 24 well! ]2 k3 x0 v2 e8 E0 G# L1 O/ g
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8. Aliquot the cell suspension into plates in the volume specified / g; \ L2 h% W! [) @8 J' [" o- l
for each plate. Remember to use Feeder plates. Always check the feeders 0 v% _4 V; M2 D, ]5 v* O
before using them. They should be confluent, no gaps, not contaminated
u' A, R2 Z. j+ j$ N; z! band not dividing. Use feeders that are older, (1-2 weeks old), the : m1 [3 v' O6 B V5 H4 @
advantages are many: any contamination is assessed, also any dividing
* @+ L2 K( K% S+ |, srun-away cells can be detected, and the passage will be earlier. Also,
% m0 P( z: {. D- m/ ?older feeders have settled nicely and flattened.4 ~' P0 Y, @& C, c; N$ `5 E
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9. Mix to have a uniform cell distribution. Return plates to the TC ) c) l7 H$ @+ U3 B( v$ ^
37 oC incubator.
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" v4 _0 v0 K0 l8 m" S/ [FREEZING ES CELLS (SLOW FREEZE)
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1. Check cells under the microscope for 80-85% confluence.
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2. Refeed cells 3 - 4 hours before passing them.3 t7 |2 e U1 N: r( U# h
3 Q' ]6 T6 p, @# q, @3. Aspirate media off. Wash one time with PBS. Add 500 祃 of ! T& W8 \/ {. v- X! @( E
trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.3 x V% ?0 C A: Z
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4. Incubate @ 37oC for 15 minutes.
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5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to
( d' Z& ^- E' [0 M6 Qinactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x , }! b" b, _" ?+ |8 [
10-cm dish." q/ K3 c' M) h' ?; r' C1 F
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6. With a transfer pipette, pipet up and down several times to ' o0 r0 r8 B* x0 f: S( h
separate the cells and break any colonies. Collect cell suspension in a
( m" z/ a- {' `- @/ vcentrifuge tube and add more media to count.
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t0 N, d% V) f6 V0 j4 }7. Count a 200 祃 aliquot and calculate the total cell number. From
( j0 D$ W4 L* q% p! u* U" u, othis, calculate the volume of media required to give a final density of , O' ?; Y5 H: t# Y
3.0 x 107 cells/ml. This density is very important, do not deviate from
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+ w: s" S7 B% s2 Z8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.
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`. S& P2 z" ?0 _2 d8 g3 g9. Aspirate off supernatant and resuspend the pellet in 1/2 the . m: k# t4 b5 s
volume calculated in Step 7 above, with M15 media.
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10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20% - D1 P3 @6 e3 z
DMSO, freshly prepared); the cell suspension is diluted as a result: 10% 5 p+ |7 h; }6 w2 {4 [
DMSO is the final conc. Add the freezing media dropwise, mixing well . O' A* f- }* P" y" |" T; ^
after each addition.
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11. Aliquot the suspension into sterile freezing vials, pre-labeled % y; _8 |( L5 P( K& L
with the cell type (AB2.2, AB1, etc.), clone number, passage number and
" S) V8 p. t5 l$ B8 o0 h# R6 y) J5 Ddate. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a
( p9 h& c8 o8 ?, Y* Kdensity = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106 9 M! e8 z4 i; ~4 h5 `8 H) d: m
cells total/vial., u2 o. u8 k! Z- t1 f- F
3 K6 j( v6 r* f* h8 I2 v, z12. Place vials into a freezing container. It is critical that the
, X, v+ E" w8 j% l Sfreezing rate is not faster than 1/minute. Do not use any untested
6 e5 C' u6 Q5 f" Jstyrofoam container since freezing rates vary greatly and this will most ; S/ f- a4 f3 E' w4 R
likely result in death of most of your cells. Freeze cells overnight at / L1 |: I; z; S. d4 o, [" o
-70oC, (24 hours).
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13. Next day, transfer cells to the Liquid Nitrogen freezer (or 0 X7 n5 q' @$ M' c$ f* ~
-135oC freezer). |
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