PROTOCOL- Fibroblast Cell

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Maintenance After Subculturing
6 B, e; Y4 i* C; G% h% gAfter 24 hours:
; f* g5 O6 Q' }9 f: z1. Examine the cells microscopically. At least 60% of the cells should have attached to the culture flask.( }6 _) R7 S* A; J3 F3 H
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Some cells will be loosely adherent, but most will have spread out on the culture flask surface. At this stage, most cells will be single or in small colonies.
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- `6 d: s. c  v& A3 H2. Change the culture medium to remove residual trypsin and non-attached cells.   c; Z# d$ N  ~" I/ w0 u8 Z3 H

4 |2 ~- o& K( ?/ T! \" Y: v& w3. Incubate for an additional 24 hours, and re-examine the culture. % e, H6 k* j/ V0 A  G. e( k& M
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a.At this stage, the vessel should have several mitotic figures indicating that the cells have resumed active growth.4 T& Z) C; H- f
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b.If few mitotic figures are observed, contact your Clonetics? Technical Specialist for assistance.
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4. Change the medium again 48 hours after the day 1 feeding, and every 48 hours thereafter while examining the culture daily. ' I3 m6 @! R7 U% p( ~

# b" J% G/ c- y; v8 `7 }0 I& O( s5. Feed with volumes as outlined in the table on page 13.. P$ I/ [6 f- T

" ?( V+ ?! n1 K' j+ [1 }  U6. Passage again when the cells are 70-90% confluent. (If seeded at the recommended seeding density, this should take 5-9 days.)
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) _7 ?4 V; z* V% F; GInstructions for Proliferating Cells. B" k/ c- l+ `) X4 n& z4 I
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Cell Preparation: Proliferating Cells
# l$ k" r: F8 @5 k% \. _* |With the proliferating culture of NHDF or NHLF you received, do the following:8 h6 h( ^& v3 R! ?0 p' T5 s; S! q8 f
% B* Y; U1 p6 G$ y! l) z$ ~' B
Examine the culture microscopically for any signs of distress during shipment (i.e., detachment, rounding-up or atypical morphology). Check the relative cell density and estimate "% confluency." The culture should be 30-80% confluent upon receipt. Some cellular detachment is normal. Please call Clonetics? Technical Specialist immediately if cells look severely distressed. : K, `; X7 O1 D- N
Decontaminate the external surface of the cell culture flask by wiping with 70% ethanol or isopropanol.
9 p3 \, z6 S6 P" w' [8 p* UIncubate the sealed flask at 37?C, 5% CO2 for three to four hours to equilibrate temperature. % z  r8 Q7 C& \0 f) D
Warm an appropriate amount of growth medium (see table on page 13) to 37?C in a sterile container. Warming the entire bottle can shorten the life of the medium. Never warm growth medium under hot running water or any other uncontrolled temperature source. NEVER MICROWAVE! , [1 k' o- A# X: K: u2 k9 Z
In a sterile field, carefully open the cell culture flask, remove the medium and replace it with the warmed, fresh growth medium. Aseptically remove any medium inside the neck or cap area because it can facilitate microbial contamination.
# b) `" M9 X/ J. \; {& C6 j# xLoosen the cap, and return the flask to the 37?C humidified incubator with 5% CO2 for at least 24 hours.

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Subculturing
0 p! B+ e" Z/ K1 q5 W) QExamine your cultures microscopically every day.
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Subculture the cells when they reach 70-90% confluency. NHDF and NHLF cultures should have many mitotic figures throughout the flask. Cells should be ready to subculture within 24 to 48 hours, however, shipping conditions such as temperature fluctuations may affect the actual time at which the cells are ready for subculture. 1 r; ]1 e) h9 F& x" v! ?  X" S+ v
Avoid the loss of your culture due to contact inhibition by subculturing cells at no more than 90% confluency. See pages 15-17 for detailed subculturing instructions.

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BIBLIOGRAPHY 2 ^- F; _9 ~6 F6 U0 C

2 v- x% E9 `9 \; Y5 X$ Z; h1) 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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2) Cytokeratin 18 & 19. Call your Clonetics? Technical Specialist for a reference on this procedure.
5 C; _/ A. ]6 ?2 L4 Q
$ P7 [( @8 e& R$ w& O3 v' s% [7 J3) Wagner, D. D., Olmsted, J.B. and V.J. Marder. (1982) Immunolocalization of Von Willebrand Protein in Weibel-Palade Bodies of Human Endothelial Cells. Journal of Cell Biology., 95:355-360., ]: d0 A/ I9 x+ `9 E6 G! Y

: K" p: z' M- l4 I+ S" v4) Grizzle, W.E., and S.S. Polt. (1988) Guidelines to Avoid Personnel Contamination By Infective Agents in Research Laboratories That Use Human Tissues, J. of Tissue Culture Methods, Vol. 11, No. 4.) V. h5 o/ j) b3 L
3 I, @+ J. B- c( b0 \7 \
TECHNICAL SERVICE:
, @* x9 Z) G" p: hBioWhittaker Inc.8 X# M  \+ {" Q" S( Q; I
Clonetics? Products$ f, b- D/ N1 }% Q; c
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' J% d, W; }0 @$ t
INTERNATIONAL TECHNICAL SERVICE:
, K  A# m5 N* z  `4 f. n
! w/ }( G* k' S8 H1 S1 w8 LBioWhittaker Inc.
) m/ N( Y! _% Q1 Q2 B8 U; ?Clonetics Products8 p3 ]9 V; M! d$ e+ {2 P6 I
8830 Biggs Ford Rd./ X) K! m/ p; {- L& t
Walkersville, MD 21793-0127
4 o" ]- Q3 w1 C$ y301-898-70252 y0 ~" Y4 J0 ], c1 A6 k8 |9 f% i; }& C* R
FAX: 301-845-2924: i- m3 r; V4 A3 c, _  k* L/ D
E-mail: techsup@biowhittaker.com8 h& d* A# B  {  M# m. O

1 M9 t9 g+ o& S2 h' `% T$ c/ JORDERS:
4 L# J6 ^4 n- @: {3 I) @1 dBioWhittaker, Inc.3 z/ Z" {+ J; D/ I" g+ I6 J9 j+ ~" `
Clonetics? Products
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APPENDIX A+ e3 Q* A7 x7 o
OVERVIEW OF FIBROBLAST MEDIA4 i/ k- L2 ^, y" e+ L

7 a/ R6 `% |- Z+ o) a. P( ^! Y500 ml Bottles (except where indicated)
+ V% @' f+ O, r' B2 w7 K/ i0 H2 g$ ?7 ^/ i
CC-3131 FBM?   Fibroblast Basal Medium (no growth factors)
; n: w, ]0 h& s0 \CC-3130 FGM? BulletKit?   Kit which contains a 500 ml bottle of FBM?, (CC-3131) and FGM? SingleQuots? (CC-4134) which contains all of the supplements listed below, conveniently packaged as single-use aliquots (amounts indicate concentration of each SingleQuot?)( E$ U( b, I- ^- Q  [- F
1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml( ^# ]- P( u, i2 j4 [, `
5 mg/ml Insulin (CC-4021) 0.5 ml5 U$ ~( p# G- C8 }. i
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml
2 D) G6 b* U; k0 e( O
% S7 w  H' }0 _2 `4 m* f! eCC-3132 FGM?-2 BulletKit?   Kit which contains a 500 ml bottle of FBM?, (CC-3131) and FGM?-2 SingleQuots? (CC-4126) which contains all of the supplements listed below, conveniently packaged as single-use aliquots (amounts indicate concentration of each SingleQuot?)
" T% @! R% I7 e1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
; _: Q5 S* n5 a' l: {5 mg/ml Insulin (CC-4021) 0.5 ml ) V4 {& d7 o2 V6 x4 \5 Y
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml 1 F; y& G/ N- }6 G, Y& k& O1 W
10 ml FBS (Fetal Bovine Serum) (CC-4101)
6 m% F6 c5 m9 \) {3 _/ Y% V( ~ * V  r9 v' t3 D/ X) {9 p
CC-3134 FGLM™ Custom   FGM? Labeling Medium BulletKit? (500 ml) that consists of the following: 0 i- g2 {; o$ n8 t, r* j4 ]. Z4 H
  CC-3133   FBLM™ Fibroblast Basal Labeling Medium, without the following nutrients: Myo- Inositol, Thymidine, Proline, Isoleucine, Leucine, Methionine, and Cysteine.
- J9 d% W, \/ C( Q! o: f  CC-4153   FGLM™ SingleQuot? Kit, FGM? labeling SingleQuots? consisting of the following:
- F& _* T7 L4 e5 W1 P8 b7 g9 U1 r- ~3.513 mg/ml L-Cysteine(CC-4069)2 ml' v: d3 [. a4 n: \4 ^
6.559 mg/ml L-Isoleucine(CC-4045) 1 ml
+ i& O2 y8 Q6 C" Q9.01 mg/ml Myo-Inositol(CC-4050) 1 ml6 k& F6 K3 X$ `6 w' G4 G
9.838 mg/ml L-Leucine(CC-4046)2 ml5 v+ X" Y9 |% q; O. E
2.238 mg/ml L-Methionine(CC-4047) 1 ml' b7 E1 t3 @1 l2 D$ B% L
2.878 mg/ml L-Proline(CC-4048)1 ml
: T, Q! ~' C" A3 w% G. O0.036 mg/ml L-Thymidine(CC-4049) 1 ml! b2 C( R. i! ^  @7 h3 [* l' Z
* r; N9 ^% V# Y- Y! S- s
  CC-4134   FGM? SingleQuots? (See CC-3130)
9 @5 |( W" e. x' ?5 \CC-3135 FGLM™-2 Custom   FGM?-2 Labeling Medium BulletKit? (500 ml) that consists of the following:
# d1 q6 v$ q( u3 G0 D9 L9 \, N1 H2 Z  CC-3133   FBLM™ Fibroblast Basal Labeling Medium, without the following nutrients: Myo- Inositol, Thymidine, Proline, Isoleucine, Leucine, Methionine, and Cysteine. 2 j, k* ~7 _/ g5 m, h0 ^
  CC-4153   FGLM™ SingleQuot? Kit, FGM? labeling SingleQuots? consisting of the following:
9 ]( o  @6 [" r% M: y1 v! g2 Q3.513 mg/ml L-Cysteine(CC-4069)2 ml: e4 B3 U! [/ ]- J0 C5 R. n, L
6.559 mg/ml L-Isoleucine(CC-4045) 1 ml" F+ U- j. `3 t' o
9.01 mg/ml Myo-Inositol(CC-4050) 1 ml
. v! I; H9 r8 d' G4 A5 W0 I9.838 mg/ml L-Leucine(CC-4046)2 ml
! F. q! q7 B  ]  ^, E3 b: N5 `! b2.238 mg/ml L-Methionine(CC-4047) 1 ml
2 v2 S0 n6 P5 P- {0 E2 o7 ]! N2.878 mg/ml L-Proline(CC-4048)1 ml3 y3 {3 V) ~+ b
0.036 mg/ml L-Thymidine(CC-4049) 1 ml8 C( @# R/ t& m) m$ B% a1 [: D

3 o6 @9 q4 e5 ^% |  CC-4126   FGM?-2 SingleQuots? (See CC-3132) : ^8 Z/ X9 {) Q9 A

; q/ F* B4 D+ LNOTE: All Clonetics? Media can be custom formulated to meet your research needs. Contact your Technical Specialist for more information.

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APPENDIX B
/ M) H4 H* o9 C, L7 F+ g' a! iCELL COUNTING USING A HEMACYTOMETER
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3 P% X" z, R" x, v+ _+ z; ~5 tBackground
$ S* F: P, h/ K9 ]5 XProper 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. 5 r$ F/ n' o" T  }) B* Q% K

0 v6 w; |8 ^  S0 w# i, [% i- YProcedure# `8 a6 a' [6 D5 d. z
1.Prepare a cell suspension as instructed in step 13 on page 16.
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- c; i2 e* n: j0 p9 `2.Prepare a hemacytometer for use. " w: c# L  n+ P3 d" ~

* p! A  b" l$ p/ n! v6 la. Carefully clean all surfaces of the hemacytometer and coverslip.
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b. Take care to ensure that all surfaces are completely dry using non-linting tissue.' x/ C: ~, F. z8 s( ?. \6 ^

, Q: q$ P" C" r) c! Hc. Center the coverslip on the hemacytometer.
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6 F7 B8 L8 S2 Q' j3.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.8 N5 k6 E1 w: _) u2 _8 H& ~( s

2 b9 J. D6 @: `( q& t. E; ^2 ]9 ^a. Use a clean pipet tip.) E- I3 r4 J. L4 a+ L  i. z% M
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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.0 H; \% s) A. |3 G: B% P

8 O, d  q' w( ~, x3 i' V2 od. Avoid injecting bubbles into the chambers.  M( \. q5 U; `& x' o" X8 J( r0 n
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e. Do not overfill or underfill the chambers.4 C! `4 P# a( ]4 r0 k

6 {* c1 d1 j  M- m! x* `4.Count the Cells.
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6 B. V4 m/ Q% T1 A! D' Ta. Allow the cell suspension to settle for at least 10 seconds.
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) p5 ?3 ?2 T0 I4 u. t: _b. 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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1) DO count the cells touching the top or left borders. % y8 P$ x% b- Z9 {3 k
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2) DO NOT count the cells touching the bottom or right borders. + |! ^% b8 o0 v0 K/ O  q

+ \, [( o9 o0 Z: x- VHemacytometer Reference Figure 1
" ~. t' y/ t) _5.Determine the Cell Count.3 ]( p! M+ T( i& [( |

2 R% f0 H$ ~9 @7 Ha. Calculate the total cells counted in the four corner squares., P0 Y* I' B0 n" j3 Z

; S+ ^6 [3 \* E4 o+ Y: [. b1) 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).
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( _- c5 Y( @; W0 L  Z+ K2) 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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9 n: y5 K5 u/ j  ^% Q4 KNOTE: If satisfactory results are not achieved, contact your Clonetics? Technical Specialist by telephoning 800-852-5663.& T; ?) Y- j' m' s1 B3 y
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b. Calculate the cell count using the equation: cells/ml = (n) x 104,
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7 o) K1 Y" N6 S8 P2 O- Fwhere: n = the average cell count per square of the four corner squares counted.
3 _. N( ^/ J9 x$ B" I  X* Y) F% g( `0 R# B- G& `
Example:If the calculated average (n) of cells in the four 1 mm corner squares of the hemacytometer is 30:5 H6 ~9 U7 D0 b1 t

' G" B& s3 f7 d/ a  X8 s( W" ~  bcells/ml = (n) x 104 (or) cells/ml = 30 x 10,000 = 300,000 cells/ml./ O) u7 l$ W) M+ M5 E! {
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c. Determine the total number of cells in the total suspension volume./ q9 S( T2 W) o& O
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1) Determine the total volume of the cell suspension.
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( J& L* h' V: b5 e) [3 {" g2) Multiply the volume of the cell suspension by the "cells/ ml" value calculated above.
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Example:If the initial suspension volume is 2 ml:
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7 t$ \; B! U; i* B/ P, n- ]cells/ml x total volume = 300,000 cells/ml x 2 ml = 600,000 cells.

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APPENDIX C 6 s7 z/ ^3 r8 c! O
ASSESSMENT OF CELL VIABILITY WITH TRYPAN BLUE
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Background
: x7 d% n: t) J/ T% S! E8 xTrypan 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.; D* }4 l8 M9 k4 D- y  x& B

# d3 j3 j! Y" \Using Trypan Blue
$ L8 U' N1 P$ z" U1. Prepare the hemacytometer for use.
9 I+ I4 x' `; f) M% j+ A
% Q7 v4 O& R+ R0 `a. Carefully clean all surfaces of the hemacytometer and cover slip./ i" c2 o: @" F$ f! [$ ~  k

+ H3 X, ]" h  e% Q2 bb. Take care to ensure that all surfaces are completely dry using non-linting tissue.
. m+ n; U; x  p" G: {# ~5 t) w$ h* g9 ~" e
c. Center the cover slip on the hemacytometer.0 t( L4 l5 U8 U/ K% f

7 i  L. ]' i9 _: ?" `. |1 n+ s% I: p2. Transfer 50 ml of 0.4% Trypan Blue into a clean tube.1 z7 n# v% b: X. d: k  O; Z3 G' V
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3. Add 50 ml of the prepared cell suspension into the tube containing the stain.
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2 H% E: @. h: N( Z9 h4. Mix the solution thoroughly, but gently. Take care to avoid making excessive bubbles.
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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.). n& l* x( _0 e, G

  v$ V5 p3 j! ?7 }: [8 l, o$ J+ i6. 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.
7 s0 y! Q5 k4 {8 b# q
& i# S( P. d9 }; D1 |" b! [% ya. Use a clean pipet tip.
* t3 o4 e2 y0 {, Q2 M* Y0 D
1 X" f+ T" I8 n4 g) ]; h( ib. Be sure that the suspension is mixed thoroughly but gently before drawing the samples.
% J' @; \6 C% v" S# e! l; b
9 z* A  l/ f" j" \3 M' ?c. Fill the chambers slowly and steadily.; G' z$ Z6 L' L$ {

+ W$ ^4 [9 s1 H  T, s1 d# Ud. Avoid injecting bubbles into the chambers.5 i$ E* g! z1 [( h+ `' h
/ P+ H3 k" w+ _7 F) D" I' }# s3 M! l
e. Do not overfill or underfill the chambers.
2 r" o- q- _, L4 Q5 I9 K# z0 z( o; z
7. Determine Cell Viability.
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a. Allow the suspension to settle in the chambers for at least 10 seconds.) O! S$ x4 n4 Z2 P8 l5 ^
! n7 f# J- f# K2 {" z) N' ?' d
b. Count all of the stained cells in each of the four corner squares of the hemacytometer.1 a( }& v# F9 s; A& S5 X

. j3 ]& x1 s/ w$ G) A5 tc. Separately count all of the unstained cells in the same squares.
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d. Calculate the cell viability using the equation:
6 e5 \" \7 K* w; }% j, A! n* a5 h6 g. A7 }5 A
% Cell Viability = number of unstained (living) cells / Total cells counted (stained + unstained) x 100%6 N* w" k" r# h; W
) n; o* k7 A  {5 E
Example: If a total of 300 cells (stained + unstained) are counted and 200 are identified as living cells (unstained), then the viability is calculated as:
4 _( v! t% L; D9 `$ l3 p  g% O8 i+ w4 \
% Cell viability =200 / 300 x 100% = 67%

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IMPROVING CELL YIELD AND VIABILITY
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Background
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! Y) K$ e' Q; C: {& NSeveral 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.
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Improving Cell Yield
! v9 T# R/ d0 {; H  ?
0 \7 }( ]: q4 g* c/ \9 p" VIf 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).
( n0 l6 @* ]* A* j1 b- l5 Q# d$ N
" P: O5 s6 [5 n* R9 V% @5 z. sLow Yield (Cell Count) 5 K8 c# z, g# i/ w9 ^! D
CONDITION POSSIBLE CAUSES SOLUTIONS
- W: C& b, L# M$ {, lMajority of cells did not detach. Inactive or cold Trypsin/EDTA.
$ a1 b' f: q7 h$ F& p! u+ b9 X+ y  BImproper storage of Trypsin/ EDTA. $ y  \* q: I5 [; Y& P- j1 d
Exposure time to Trypsin/EDTA was too short.
* D, W2 m0 C; Y' c: q$ N( ~( rTrypsin/EDTA is neutralized. 1 N- P+ ^3 W! W# g. |4 l
Vessel was not "rapped" enough during trypsinization. ' J0 b6 ?3 M. N/ u5 T; N" X0 w
Use Trypsin/EDTA at room temperature. 5 W+ a- T* g( Z1 S/ y1 E
Store at -20?C until ready for use; thaw and allow it to come to room temperature briefly before subculturing.
$ Q9 G: O, c; w5 h: OIncrease exposure time to Trypsin/ EDTA. (see pg. 16)
0 e. ?" q) V8 ]5 t! J" C8 UBe sure to rinse the culture completely with HEPES-BSS before trypsinization.
: o. |) W* F- ]! EUse a moderate amount of force when rapping (see page 16).
7 J  ^- V2 u* {) S  _* [ " }5 E! ?, `- ]4 ~8 A
95% 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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Improving Cell Viability
* j: z0 _9 l2 e" YIf 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).
$ M0 X9 K! G: T7 R, W
" `) N) z# Q7 q& ULow Viability (Live Cells vs. Dead Cells)
9 m5 r, [5 x: W; l4 D6 BCONDITION POSSIBLE CAUSES SOLUTIONS
, F4 R7 r( ~0 M& a1 nTrypsin/EDTA damaged the cells. Trypsin/EDTA used at the wrong concentration.
) }6 ]/ i1 n; T( ^' |) [Exposure time of the cells to Trypsin/EDTA was too long.
0 _7 Z& A4 x8 |7 Q/ yTrypsin/EDTA was used above room temperature. Trypsin becomes more active at temperatures above room temperature.
1 w3 B$ s  `8 g4 p* A* w* I# Y, GFailed to neutralize the trypsin. Prolonged exposure to trypsin will damage the cells.
) A: }/ u( E# wVessel was "rapped" too firmly (see pg. 16) during trypsinization. Rapping too hard to release cells causes cell membranes to tear apart.
7 l: S+ j0 W3 J# f, ]( W5 h3 P8 F We recommend a trypsin concentration of 0.025% and an EDTA concentration of 0.01%.
5 k1 P4 ~7 H' x" B& s! BDo not trypsinize longer than 7 minutes.
" [7 ?. _; Z" QDO NOT USE EVEN MILDLY HEATED Trypsin/EDTA.
3 k8 H; j& c9 F3 FNeutralize the T/E with TNS to eliminate cell damage due to trypsin.
# X) C) U3 A! V: QUse moderate force when rapping.
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2 ^5 B. U+ U5 A0 S7 p% c, h# d% fCulture 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.
: {: s. w6 ^3 E6 t/ G  xCell 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.
( t7 W* ~4 c5 a7 {! {2 N3 g9 m
: q$ [- `7 N! f- M, N+ _" f! yOnce you have determined how to achieve high yield and high viability, subculture the remaining flasks.

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APPENDIX E
4 s) ?9 C) T& W7 ?* P5 MGROWTH AREA OF COMMON PLASTICWARE

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APPENDIX F # z% B/ o2 B9 _! v. E
Seeding Into Multi-Well Plates, ^6 V1 G$ J$ `! U

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0 M. q9 L- }% z, }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.  ]( l1 o7 N  Q- a
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Required Materials:3 F. n4 Z; H2 c  z* Y, g

* y0 X2 ]: X3 {( J6 E) O% T' `T-25 flask of proliferating normal human cells between 70% and 90% confluence. " ^4 e5 U+ V* x2 S
Flat-bottom, Multi-well tissue culture plates 8 n, _% l# F+ ?
37oC humidified incubator with 5% CO2 /95% air ' U9 p" `7 Q8 D" M* p  u2 P2 b
Laminar flow hood or other sterile environment
& U* X+ b' q5 d+ sAdjustable multichannel pipetter (8- or 12-channel) or repeating pipetter " o1 w, r7 x% Q2 l2 v8 I$ m
Sterile reservoir(s) for use with multichannel pipetter 7 o. b5 i$ ^7 Y
Procedure, a$ L+ Q' H  J
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Follow the steps on pages 13-15 for subculture preparation and subculturing. Then follow steps 2-4 below.
/ g3 s+ g7 O" ]; B- D! z: ~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.
" v  G( R" n8 |, b. I! J9 STransfer 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.
$ K0 {, J% u2 a# }, s3 ~# o( ]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 B/ G) B, n' r5 \, y& s5 DNOTE: 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.)