ü˻ þ      

Media Preparation

NUTRIENT MIXTURE F-10

ü 1 liter Ǹ ddH20 300 ml, и Ѻ, NaHC03 1.2g, lN HCl 0.6 ml ־ ȥѴ. ٽ ü 1L ddH20 ÷ϰ, magnetic bar stirring и δ. Disposable filter Ͽ ѹ 500ml Ų. 500 ml Ű кϰ, , , δ. Ϻ(2ml) տ θ Ȯϱ ׹ thioglycolate media ־ 10ϰ 37oC Ѵ.


RPMI 1640

ü 1 liter Ǹ
ddH20 300 ml, powered media(1L), NaHC03 2.0g, lN HCl 4.4 ml ְ ش. ٽ ü 1L ddH20 ÷ϰ, magnetic bar stirring и δ. Disposable filter Ͽ ѹ 500ml Ų 500 ml Ű кϰ, , , δ. Ϻ(2ml) տ θ Ȯϱ ׹ thioglycolate media ־ 10ϰ 37oC Ѵ.

Solutions

Acetic acid (4%): 2 ml Glacial Acetic acid in 48 ml dH2O, prepared fresh.
Bromodeoxyuridine working sol: 500 mcg/ml RPMI. Sterile filter, store in dark for 1-2 wks.
Colcemid working sol: 10 mg Colcemid in 250 ml dH2O, sterile filtered, store at 4oC.

Collagenase

    Stock solution: add 16.5ml complete Chang/F-10 to a 100mg collagenase bottle, dissolve (6mg/ml). Aliquot into microcentrifuge tubes to store at -20°C for up to 6 months.

Working solution: add 0.5ml to 1.0ml thawed collagenase stock solution to 2.5ml complete Chang/F-10 media. Filter, using a 3ml syringe and an Acrodisc filter, into a sterile 15ml conical centrifuge tube.

Complete CHANG/F10 media: 45ml Chang basal media B, 42ml Nutrient Mix F- 10, 5ml Chang supplement A, 8ml Fetal calf serum, 1ml Penicillin/Streptomycin, solution, 1.1ml L-Glutamine solution, store at 4°C.
Diepoxybutane working solution (DEB): Add 5 mcl pure DEB to 5 ml sterile dH2O, mix thoroughly. Add 10 mcl of the first solution to 1 ml sterile dH2O, mix thoroughly (11 ug/ml).
Ethidium Bromide working solution: 2 mg/ml Ethidium Bromide in RPMI-1640, stored in dark.
Fixative: 3:1 Methanol to Glacial Acetic acid
FUDR (5-Fluoro-deoxy-uridine): 2.46 mcg/ml dH2O, sterile filter, store at 4 °C. To make a 0.1 mcMolar soln add 0.1 ml to 10 ml of culture media.
Gurr Buffer: Dissolve 1 Gurr buffer tablet pH 6.8 (1 liter solution in 1 liter dH2O.
Hoechst 33258 solution: 1.0 mcg/ml PBS. Protect from light, store at 4 °C for 1 week.
Hypotonic solution - KCl: 0.075 M KCl: 2.8 g Potassium chloride dissolved in 500 ml dH2O.

Hypotonic solution - sodium citrate: 0.8%NaCitrate/0.075 M KCl: 1.6g NaCitrate, 1.68g Potassium Chloride dissolved in 500ml dH2O.
Isotonic Buffer: Dissolve 36 grams NaCl, 8 Gurr buffer tablets pH6.8 (100 ml solution), and 4 Gurr Buffer tablets pH 7.0 (1 liter solution) in 3950 ml dH2O. Adjust pH to 7.0 with 2.5 ml 2M NaOH. Bring volume to 4 liters with dH2O.
Leishman Stain: Dissolve 1 gram Leishman stain in 500 ml Methanol. Incubate at 37oC overnight, filter through Whatman paper qualitative grade 1.
McIlvaines Buffer pH 7.2: 6.5ml 0.1M Citrate + 43.5ml 0.2M Na2HPO4.
Mitomycin C Stock solution (MMC): Reconstitute 2 mg vial of MMC with 4 ml sterile dH2O. Dilute 1 ml in 24 ml sterile dH2O (20 ug/ml). Dilute 1 ml of new solution in 9 ml sterile dH2O (2 ug/ml).
Thymidine Working solution: 30 mg/ml Thymidine in RPMI-1640 media, store frozen.
1X Trypsin EDTA solution; 10 ml stock trypsin solution (10X trypsin in saline), 0.1 g EDTA (disodium salt), dissolved in 490 ml Hank's Balanced Salt Solution. Sterile filtered, 50 ml aliquots. Store frozen.
Trypsin working solution (G-bands): Dissolve 0.1 gram Trypsin 1:250 in 4 ml sterile dH2O. Store frozen.


Reagents

Chang media: IRVINE cat# T100-018, 100 ml bottle, store supplement frozen, basal media at 4°C.
Colcemid: GIBCO cat# 15210-040. 10µg/ml in Hank's Balanced Salt Solution, 10ml bottle, store at 4°C.
Colcemid: GIBCO cat# 890-3014. 10 mg bottle.

Collagenase: SIGMA cat# C-9263, 100mg bottle.
EDTA: SIGMA cat# ED2SS. Ethylenediaminetetraacetic acid, disodium salt, dihydrate, 100 g bottle.
Diepoxybutane (DEB): SIGMA cat# D 7019. 1 ml bottle. Store at 4 °C.
Ethidium Bromide: SIGMA cat# E-8751. 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide, 250 mg bottle.
Fetal calf serum: HYCLONE,INC. cat# A-1111-D. Defined fetal bovine serum, 100ml bottle, store frozen.
Fetal calf serum: GIBCO cat# 200-6140. Mycoplasma tested, virus screened, 500 ml bottle, store frozen.
FuDr: SIGMA cat # F0503
Glacial Acetic acid: FISHER cat#A38-500. Acetic acid, Glacial ACS (Aldehyde free), 500 ml bottle.
L-Glutamine: GIBCO cat# 25030-032. L-Glutamine solution 100X (200mM), 20ml bottle, store frozen.
Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33193-2P. Buffer Tablets pH 6.8 (1 liter of solution)
Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33193-2D. Buffer Tablets pH 6.8 (100 ml of solution)
Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33200-2U Buffer Tablets pH 7.0 (1 liter of solution)
Hank's Balanced Salt Solution: GIBCO cat# 14170-112. HBSS without CaCl2, MgCl2, MgSO4, 500ml bottle, Store at 4 °C.

Methanol: FISHER SCIENTIFIC cat# A412-500. Methyl alcohol anhydrous (ACS)(Absolute) Acetone free, 500ml bottle.

Mitomycin C (MMC): SIGMA cat# M 0503. 2 mg
Nutrient Mix F-10: GIBCO cat# 81200-040. Nutrient Mixture F-10 (HAM) powdered form, 10x1 liter package.
Penicillin/Streptomycin: GIBCO cat# 15140-031. Penicillin/Streptomycin solution, 10,000 units/ml; 10,000µg/ml, 20ml bottle
Potassium Chloride: UW-STORES. cat# 2485 ACS granular, 500g bottle.
RPMI-1640: GIBCO cat# 31800-022 RPMI-1640 powdered form, preparedto instructions, 10 x 1 liter package.

Sodium Citrate: UW-Stores, cat# 2588. Crystal, ACS, Na3C6H507.2H20, 500g bottle.
Thymidine: SIGMA cat# T-9250, 5 g
Trypsin (G-banding): GIBCO cat# 27250-042. Trypsin 1:250, 25 gram/bottle.
Trypsin Stock Solution(1X): GIBCO cat# 25300-013. Trypsin 2.5% (10X) in saline. Store frozen.

 

CELL CULTURE PRODUCT  (̱ þడ)

Product

Unit

Vendor

Cat. No.

Price

ANTIBIOTICS

Gentamicin, 50 mg/ml

10 ml

cellgro

MT30-005-LR

$11.64

Penicillin - Streptomycin, 100X

100 ml

Sigma

P4333

$10.00

Gibco/BRL

15140-122

$10.34

Penicillin - Streptomycin, 100X

(Hybri-Max)

100 ml

Sigma

P7539

$11.00

Penicillin-Streptomycin-Neomycin

20 ml

Sigma

P4083

$7.00

 

MEDIA

DMEM, 1 g/L glucose

500 ml

Sigma

D6046

$3.90

Gibco/BRL

11885-092

$4.60

DMEM, 4.5 g/L glucose

500 ml

Sigma

D5671

$3.90

Gibco/BRL

11965-118

$4.50

DMEM/F-12 w/15 mM HEPES

500 ml

Sigma

D6421

$3.90

Gibco/BRL

11330-032

$6.25

EMEM, 2.2 g/L NaHCO3

500 ml

Sigma

M4655

$3.90

Gibco/BRL

11095-098

$4.50

EMEM, 0.85 g/L NaHCO3

500ml

Sigma

M2414

$3.90

F-10 Nutrient Mixture

500 ml

Sigma

N6013

$3.90

Gibco/BRL

11550-043

$6.25

F-12 Nutrient Mixture

500 ml

Sigma

N4888

$3.90

Gibco/BRL

11765-062

$5.50

FISCHER'S Media

500 ml

Gibco/BRL

11475-027

$13.75

GRACE'S

500 ml

Gibco/BRL

11590-056

$14.35

ISCOVE'S

500 ml

Gibco/BRL

12440-053

$11.88

McCOY'S 5A Media

500 ml

cellgro

MT10-050-LV

$6.60

MEDIA 199

500 ml

Sigma

M2154

$3.90

Gibco/BRL

11150-059

$6.25

RPMI 1640

500 ml

Sigma

R5507

$3.90

Gibco/BRL

11875-119

$4.50

 

MEDIA SUPPLEMENTS

L-GLUTAMINE 200 mM

20 ml

Sigma

G7513

$2.70

L-GLUTAMINE 200 mM

100 ml

Sigma

G7513

$11.16

NON-ESSENTIAL AMINO ACIDS, 100X

100 ml

Sigma

M7145

$8.50

SODIUM BICARBONATE (7.5%)

500 ml

Sigma

S8761

$10.00

 

MISCELLANEOUS SOLUTIONS

HEPES BUFFER, 1M

100 ml

Sigma

H0887

$15.00

METHYL CELLULOSE (1%)

250 ml

TCCF

TCCF-T-00003

$6.61

TRYPSIN IX

500 ml

Sigma

T4424

$10.25

TRYPSIN-EDTA

500 ml

Sigma

T4049

$10.25

 

SALT SOLUTIONS

HANKS Balanced Salts

500 ml

Sigma

H9269

$3.60

D-PBS (w/Ca2+ & Mg2+)

500 ml

Sigma

D8662

$3.60

Gibco/BRL

14040-182

$4.25

D-PBS (wo/Ca2+ & Mg2+)

500 ml

Sigma

D8537

$3.60

Gibco/BRL

14190-250

$4.25

 

SERUM

FETAL BOVINE SERUM

500 ml

Gibco/BRL

26140-079

$108.00

HORSE SERUM

100 ml

Gibco/BRL

16050-015

$10.00

NEWBORN CALF SERUM

500 ml

Gibco/BRL

16010-159

$32.00

Flow

29-121-54R

$31.69

 

STERILE BOTTLES

ROUND BOTTLE

500 ml

TCCF

TCCF-T-00004

$5.10

6 oz. SANIGLASS BOTTLE

140 ml

TCCF

TCCF-T-00005

$2.59

 

UNSTERILE BOTTLES

ROUND BOTTLE

500 ml

TCCF

TCCF-T-00009

$3.95

6 oz. SANIGLASS BOTTLE

140 ml

TCCF

TCCF-T-00010

$2.23

 

CELL CULTURES

CELLS

1 million

TCCF

TCCF-T-00006

$1.50

TRANSFER MEDIA

1 ml

TCCF

TCCF-T-00007

$0.16

 

LIQUID NITROGEN STORAGE

ONE BOX (100 VIALS)

1 month

TCCF

TCCF-T-00008

$4.69

[Reference]  http://www.utmb.edu/avpr/tccf/life.htm