PROTOCOL- Fibroblast Cell

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1 {/ v5 u5 s( N0 I2 [0 YTHESE INSTRUCTIONS APPLY TO ORDERS CONTAINING THE FOLLOWING CELL PRODUCTS
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Cryopreserved Cells (Single donor)
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CC-2511 NHDF -Ad   3 500,000 cells/cryovial
: A( Y/ ]# V( \& ACC-2509 NHDF -Neo   3 500,000 cells/cryovial $ E5 R# P0 v, _
CC-2512 NHLF   3 500,000 cells/cryovial
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2 Y, c1 S3 `; D6 V3 P6 H- S  v3 gProliferating Cells 4 x0 J/ @$ e+ ^8 k3 P
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CC-2611 NHDF -Ad T-25 flask    Yield: 500,000 cells ! N3 K" d5 g! J- V
CC-0252 NHDF -Ad T-75 flask    Yield: 1,500,000 cells
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CC-2609 NHDF -Neo T-25 flask    Yield: 500,000 cells - \/ y) S' F2 X- Q* q( d
CC-0210 NHDF -Neo T-75 flask    Yield: 1,500,000 cells
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CC-2612 NHLF T-25 Flask    Yield: 1,000,000 cells 1 `1 @3 @5 I3 q4 R
CC-0282 NHLF T-75 Flask    Yield: 3,000,000 cells

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谢谢！~~~

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FROM:http://www.pharma.ethz.ch/instit ... rotocols/fibro.html

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(1)Polymerase Chain Reaction (PCR) technology is covered by U.S. Patents 4,683,195, 4,683,202, and 4,965,188 owned by Hoffman La-Roche, Inc.

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APPENDIX F
% f2 [# Z$ D$ B) U! ASeeding Into Multi-Well Plates
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0 G1 `4 _7 |1 r  i: N9 yOverview
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A culture flask of normal human cells is harvested by trypsinization and subsequent trypsin inhibitor treatment. The cells are centrifuged, resuspended in growth medium and counted. The desired number of cells is then added to wells of sterile Multi-well tissue culture plates. The plates are incubated in a 37oC, 5% CO2 humidified incubator for one to three days to allow for cell adherence and growth. Seeding densities will vary somewhat with your experimental requirements. We recommend a density for Dermal Fibroblasts and Lung Fibroblasts of 10,000 cells/cm2 for all multiwell plates.
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Required Materials:& k3 T: f. s$ }' k: u
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T-25 flask of proliferating normal human cells between 70% and 90% confluence.
9 }. Q# a/ f# l( L+ ~  KFlat-bottom, Multi-well tissue culture plates
- g( @5 \2 w# T  ~& B% ?37oC humidified incubator with 5% CO2 /95% air & f0 y6 I, U; W7 A, l) n
Laminar flow hood or other sterile environment
( _9 R  D# V5 N/ E- R: U, T  M. DAdjustable multichannel pipetter (8- or 12-channel) or repeating pipetter 9 H5 U; Y  _+ N; `" C2 m6 ^; {; X) \
Sterile reservoir(s) for use with multichannel pipetter
& [! P9 }! {& v; P2 l7 tProcedure0 Y8 A+ C% l2 D; I0 G3 i
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Follow the steps on pages 13-15 for subculture preparation and subculturing. Then follow steps 2-4 below.   L& d+ k+ O( d7 I- t+ e
Since the cells/ml calculation computed on p. 20 is "per ml", one must increase the cell concentration by 4 times before seeding 96 well plates (to accommodate the 1:4 dilution when adding 250 ul of suspended cells per well). When making the cell suspension, adjust the cell concentration with growth medium. 7 D6 q7 {) N; o
Transfer the diluted cell suspension to a sterile reservoir. Using a multichannel (8- or 12-channel) pipetter equipped with sterile pipette tips, add 250 ml of the diluted cell suspension to each well of the labeled 96- well, flat bottom, tissue culture plate(s). RESUSPEND THE CELL SUSPENSION OFTEN DURING THE SEEDING PROCEDURE TO ENSURE A UNIFORM NUMBER AND DISTRIBUTION OF CELLS INTO EACH WELL BY PIPETTING UP AND DOWN A FEW TIMES BETWEEN EVERY OTHER DISPENSING. * N9 _2 i9 n# x8 `5 t
Cover and incubate the plates for 1 to 3 days at 37oC/5% CO2. (Incubation periods exceeding 3 days are generally not recommended because of evaporation of medium from the edge wells of the plate.)
9 k6 _% D3 ^: j. YNOTE: Before using the Multi-well plate culture in a bioassay, examine them microscopically for the presence of mitotic figures as a confirmation that the cells have resumed active growth. (Does not apply for all end-user assays.)

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APPENDIX E
% Q, o, i( x! nGROWTH AREA OF COMMON PLASTICWARE

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Improving Cell Viability
& I- Z5 G& i+ i$ I0 s2 Y" mIf your cell viability is low (less than 50%), determine the possible cause(s) and solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).
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, N, r0 E  |. ~: e  _  V8 fLow Viability (Live Cells vs. Dead Cells)
! x( E& f8 X# \; [; ^7 H$ PCONDITION POSSIBLE CAUSES SOLUTIONS + G: U0 S: a# T1 Q; @
Trypsin/EDTA damaged the cells. Trypsin/EDTA used at the wrong concentration.
) R) S1 ?' B1 Y0 wExposure time of the cells to Trypsin/EDTA was too long. * F$ d1 x* r3 R4 d# P# G7 c
Trypsin/EDTA was used above room temperature. Trypsin becomes more active at temperatures above room temperature.
7 ?1 B5 ~& }8 yFailed to neutralize the trypsin. Prolonged exposure to trypsin will damage the cells.
: k9 s) u0 X4 j! p6 }0 E, ]! CVessel was "rapped" too firmly (see pg. 16) during trypsinization. Rapping too hard to release cells causes cell membranes to tear apart.
- x- n" Y/ ~% g1 H' \4 k1 j We recommend a trypsin concentration of 0.025% and an EDTA concentration of 0.01%.
3 y) Q5 G* W( k( k! e! VDo not trypsinize longer than 7 minutes. 1 Y, T8 H, l/ V) M- s( T( F: e
DO NOT USE EVEN MILDLY HEATED Trypsin/EDTA. 8 }& ~# G6 f# B" J4 g' o% o
Neutralize the T/E with TNS to eliminate cell damage due to trypsin.
4 [- S' z: F! m. tUse moderate force when rapping.
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Culture vessel was too confluent. Culture was too confluent at trypsinization.  Be sure to trypsinize at 70-90% confluence with about five mitotic figures per field of view. " o2 f3 ~7 D( u
Cell growth slowed before 90% confluence and cells look dull and non- refractile. The most probable cause is failure to increase the volume of medium used as the cell confluency increased. The cells become mildly starved and are not able to recover after trypsinization.  Change medium and increase volume as recommended. Please observe all guidelines.
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& @0 Y4 o2 e: ^+ UOnce you have determined how to achieve high yield and high viability, subculture the remaining flasks.

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IMPROVING CELL YIELD AND VIABILITY! ?& k2 b  b" [
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Background) _. Q3 `% C/ u. C) P6 {! V

0 Y. b3 m3 L- X% A* a# PSeveral factors, or a combination of factors, contribute to low cell count and low cell viability. If cell yield or viability is unsatisfactory, use the following information to increase the success rate of future cultures.6 K% w0 q8 {+ ?7 h' Z8 M

1 `/ P' I5 h/ H0 F4 MImproving Cell Yield . B  G4 N6 G- A1 f2 T5 o5 [$ j
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If your cell yield is low (less than 50%), determine the cause(s) and possible solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).
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Low Yield (Cell Count) ' j6 ?. p1 G4 u+ U# u) ~9 ~4 _$ c
CONDITION POSSIBLE CAUSES SOLUTIONS , v0 w0 P1 j% d- \/ P
Majority of cells did not detach. Inactive or cold Trypsin/EDTA.
* G: b+ m5 ?6 C4 iImproper storage of Trypsin/ EDTA.
& t& k, f5 _% @& P! i2 u' d9 G6 UExposure time to Trypsin/EDTA was too short. 9 O9 l; S2 l! E8 }4 {) V  J4 {1 V" b
Trypsin/EDTA is neutralized.
6 W, V& ^/ J* f+ L8 s! IVessel was not "rapped" enough during trypsinization. , A: K% x. Q0 _% ^$ w
Use Trypsin/EDTA at room temperature. 5 q) e3 |; `. E9 X# k, R
Store at -20?C until ready for use; thaw and allow it to come to room temperature briefly before subculturing.
- p8 [) Q) k* A5 `1 TIncrease exposure time to Trypsin/ EDTA. (see pg. 16) ( ^8 R( }) D) O$ H5 {
Be sure to rinse the culture completely with HEPES-BSS before trypsinization. ' s; M4 t( n5 s/ Q
Use a moderate amount of force when rapping (see page 16).
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, {+ O, [6 U' _! O! S. H95% of the cells detached but the yield was low. Culture was under confluent at trypsinization. Be sure to trypsinize at 70-90% confluence with at least 5 mitotic figures per field of view.

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APPENDIX C " r1 s9 K6 x9 N0 p
ASSESSMENT OF CELL VIABILITY WITH TRYPAN BLUE
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: f8 _' a  d# q6 e5 `7 E  BBackground
9 W1 v% Y$ H. n6 x% k. T( M# d. JTrypan blue is a dye that enables easy identification of dead cells. Dead cells take up the dye and appear blue with uneven cell membranes. By contrast, living cells repel the dye and appear refractile and colorless.
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Using Trypan Blue
6 x% S' F+ P' \+ \1. Prepare the hemacytometer for use. " x  W5 n1 ^: a) q
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a. Carefully clean all surfaces of the hemacytometer and cover slip.
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b. Take care to ensure that all surfaces are completely dry using non-linting tissue.2 |8 X* a4 \& W, c" q

7 r; D) V- J, ^8 e  D) v6 Mc. Center the cover slip on the hemacytometer.
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" m0 r- z; d$ d9 t4 K0 Z* @% g2. Transfer 50 ml of 0.4% Trypan Blue into a clean tube.* J* f8 K/ R1 d  B, ]

( v7 x2 W' g) H7 X0 e" I; B3 M& A# e3. Add 50 ml of the prepared cell suspension into the tube containing the stain.. c$ @" V) I! @4 C# \# U) T, _. Y
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4. Mix the solution thoroughly, but gently. Take care to avoid making excessive bubbles.# J; E' N2 O; ~, s! t5 k8 K/ D
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5. Allow the mixture to sit for 2-3 minutes after mixing. (Do not let the cells sit in the dye for more than five minutes because both the living and dead cells will begin to take-up the dye after five minutes.)2 T# L6 C  W. N* ~' |2 l# N+ h/ G+ ]

$ I( ^3 Y& |( I% ^) \, B! {6. Pipet approximately 9 microliters of the Trypan Blue/cell suspension mixture (this volume will vary with brand of hemacytometer) into one of the two counting chambers.
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a. Use a clean pipet tip.- T' Q) k4 ~/ M  N
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b. Be sure that the suspension is mixed thoroughly but gently before drawing the samples.# G: A8 _1 Y: o8 F) T
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c. Fill the chambers slowly and steadily.
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d. Avoid injecting bubbles into the chambers.
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e. Do not overfill or underfill the chambers.
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7. Determine Cell Viability.
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a. Allow the suspension to settle in the chambers for at least 10 seconds.
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b. Count all of the stained cells in each of the four corner squares of the hemacytometer.
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c. Separately count all of the unstained cells in the same squares.3 e/ t+ N+ y! Y
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d. Calculate the cell viability using the equation:! m' x# ^5 o; k# l. \6 R
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% Cell Viability = number of unstained (living) cells / Total cells counted (stained + unstained) x 100%1 Y- Q. u# I) x9 p2 E' b% [

" }# _, x9 w+ O! T- K) C3 H( qExample: If a total of 300 cells (stained + unstained) are counted and 200 are identified as living cells (unstained), then the viability is calculated as:- F7 U9 w; \/ E' B
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% Cell viability =200 / 300 x 100% = 67%

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APPENDIX B 5 f% c# C9 Q; {2 [& a
CELL COUNTING USING A HEMACYTOMETER/ t) U7 |! K/ N* @

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Proper use of a hemacytometer is critical for obtaining an accurate count of cells and is a procedure used by BioWhittaker to determine the suspension counts for Clonetics? cell strains. A hemacytometer consists of a thickened glass slide into which a small chamber has been cut to allow for the introduction of cells to be counted. The floor of the chamber is divided (etched) into nine sections; usually only the four corner sections are used in cell counting (See Figure 1 below). With a coverslip in place, each square of the hemacytometer represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is approximately equivalent to 1 ml, the cell concentration per ml (and the total number of cells) can be determined.
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2 P" M3 g$ J' B! s3 DProcedure3 `. q1 x( b6 G8 A8 ^" c# ^, m+ L! r& P4 _
1.Prepare a cell suspension as instructed in step 13 on page 16. , m1 r( B! l* m6 E, M3 s

3 [$ b) J# K( d) G" h2.Prepare a hemacytometer for use. * G7 g" r! l: o5 A& X5 y/ H' W
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a. Carefully clean all surfaces of the hemacytometer and coverslip.
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" M. B; e) t0 X: k  l. B# fb. Take care to ensure that all surfaces are completely dry using non-linting tissue.
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c. Center the coverslip on the hemacytometer.
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- [- D8 J6 U6 j7 D, T; Y0 q8 m, t" q3.Pipet approximately 9 microliters (this volume will vary slighting with the brand of hemacytometer) of the cell suspension into one of the two counting chambers.. s- z3 a2 }0 ^* P) o  Q( `/ g' m: U
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a. Use a clean pipet tip.
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% _1 h. b5 F2 d# Y# u. ^b. Be sure that the suspension is thoroughly, but gently, mixed before drawing the samples.
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c. Fill the chambers slowly and steadily.
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d. Avoid injecting bubbles into the chambers.& k9 M; Q6 @$ I6 J7 b

. H: F* S! w/ ie. Do not overfill or underfill the chambers.
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7 ^2 f8 E# S6 ~: i2 J4.Count the Cells.
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5 S8 [9 s9 H7 ja. Allow the cell suspension to settle for at least 10 seconds.
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. h  v, H0 s( ^( C* |. ]( g9 kb. Count all of the cells in each of the four 1 mm3 corner squares labeled A thru D in Figure 1 on the next page.
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8 r/ [! L/ O2 B; u1) DO count the cells touching the top or left borders. ; P' M1 z& G1 t+ [* H6 H0 U
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2) DO NOT count the cells touching the bottom or right borders.
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2 [( E% B4 n. g6 W" D  HHemacytometer Reference Figure 1
- P  r0 j) H. ~+ x5.Determine the Cell Count.6 U( @- m7 b; Y) D

7 L5 ?, c* x7 N4 Y+ q$ Y) N8 i, Oa. Calculate the total cells counted in the four corner squares.3 F: F* ^4 y! @, ]! _

  w. r" h2 y0 [3 m5 @. k1) If the total cell count is less than 100, or if more than 10% of the cells counted appear to be clustered, carefully re-mix the original cell suspension and repeat steps 2 through 4 (above).& j# S, C* c6 R0 A8 q

  m1 n2 q/ y1 h* \% s/ A2) If the total cell count is greater than 400, dilute the suspension so the count will be 100-400 cells. Then repeat Steps 2 -4 (above).
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NOTE: If satisfactory results are not achieved, contact your Clonetics? Technical Specialist by telephoning 800-852-5663.. f4 R2 H$ J. U- m8 j% }! u! u
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b. Calculate the cell count using the equation: cells/ml = (n) x 104,
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: A7 {/ a" p$ m# E+ lwhere: n = the average cell count per square of the four corner squares counted. 6 d: `1 W5 C5 P8 d

* l  l4 w5 K/ ~0 Q" y. q! L$ o: JExample:If the calculated average (n) of cells in the four 1 mm corner squares of the hemacytometer is 30:
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cells/ml = (n) x 104 (or) cells/ml = 30 x 10,000 = 300,000 cells/ml.
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c. Determine the total number of cells in the total suspension volume.
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! t0 {8 L; }* p1) Determine the total volume of the cell suspension.
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, n" c7 n  k5 m/ f2) Multiply the volume of the cell suspension by the "cells/ ml" value calculated above.
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; V9 l: j& Q/ T1 I8 ZExample:If the initial suspension volume is 2 ml:+ r9 f; T: X6 t% ], {( X7 S# M& ^; L' z
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cells/ml x total volume = 300,000 cells/ml x 2 ml = 600,000 cells.