TISSUE DESINTEGRATION ENZYMES

| Biotase | Collagenase | CLS I | CLS II | CLS III | CLS IV | Trypsin-EDTA | Papain |

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BIOTASE

Now available from Biochrom under the trade Biotase“, Biochrom offers an enzyme equivalent to Accutase^®. The product is meant for the detachment of cells with a more sensitive detachment profile. As the raw substance for Biotase is neither of vertrebrate, nor bacterial nature, the risk of virus contamination, and endotoxin load are excluded. Biotase will detach anchorage dependent cells within very short time, leaving the proteins of the cell matrix intact. Biochrom offers Biotase, solved in PBS, in a 100 ml bottle. Storage condition of the product is -20°C; shelf life 12 months. Biotase is now available under the product code L 2193. Once thawed, Biotase will remain active at +2 -+8°C for at least 7 days. Avoid repeated thawing cycles; rather aliquote, and deep-freeze, according to demand.

	Cat. No.	Unit
Biotase	L2193	100 ml

COLLAGENASE

Storage: +2 - +8⁰C

Collagenase from Clostridium histolyticum

Cells of animal tissue are attached to each other by a complex matrix of proteins, glycoproteins, lipides, glycolipides, and mucopolysaccharides. To isolate individual cells or to start primary cultures, the delicate matrix must be partially broken down without destroying the cell or its surface.

Collagenase are the enzymes of choice for this application, if dissolved in balanced salts.

Actual enzyme concentrations should be 0.1 to 0.2 % (w/v), depending on the delicate nature of the tissue substrate.

Definitions:

Collagenase activity according to Mandl: 1 unit releases 1 μmol amino acids with ninhydrin, determined, and calculated as leucin, from native collagen with-in 5 hours (37^oC and pH 7.5).

Collagenase activity according to Wünsch and Heidrich: 1 PZ-unit catalyses the hydrolysis of 1 pmol of the water soluble chromepeptide PZ-Pro-Leu-Gly-Pro-D-Arg per minute (25^oC/pH 7.1). Resulting PZI-L-proline (PZ = A-Plnenyl-azobenzyloxy-carbonyl) can be determined.

Reference: Tissue Culture, Methods and Applications (ed. P.F. Kruse and M.K. Patterson) Academic Press, New York [1972]

CLS Type I:

Enzyme blending from collagenase, clostripain, with tryptic and proteolytic activities, from Clostridium histolyticum. This preparation is recommended for liver, lung, adipose, and adrenal tissue. Specific activity is 125 to 250 Mandl units per miligram of powdered substance.

	Cat. No.	Unit
Collagenase	C1-28	100 mg
type I, CLS	C1-22	1 g

CLS Type II:

Enzyme blending from collagenase, clostripain, with tryptic and proteolytic activities, from Clostridium histolyticum. This preparation shows a high clostripain activity with the tryptic activity close to type I. Suitable for liver, bone, thyroid, heart, and salivary gland tissue. Specific activity is 125 to 250 Mandl units per milligram of powdered substance.

	Cat. No.	Unit
Collagenase	C2-28	100 mg
type II, CLS II	C2-22	1 g

CLS Type III:

Enzyme blending from collagenase, clostripain, with tryptic and proteolytic activities, from Clostridium histolyticum This preparation has a very low proteolytic and a normal collogenase activity. Suitable for mammary gland tissue. Specific activity is 125 to 250 Mondl units per milligram of powdered substance.

	Cat. No.	Unit
Collagenase	C3-28	100 mg
type III, CLS III	C3-22	1 g

CLS Type IV:

Enzyme blending from collagenase, clostripain, with tryptic and proteolytic activities. Recommended for the isolation of Langerhans' islands cells. Specific activity is 125 to 250 Mandl units per milligram of powdered substance.

	Cat. No.	Unit
Collagenase	C4-28	100 mg
type IV, CLS IV	C4-22	1 g

TRYPSIN / EDTA

Storage:-20⁰C.

Based on crude trypsin derived from porcine pancreas; loss on drying: ≤ 5%. Specific activity range: 250-300 USP U/mg.

Unit definition: 1USP unit catalyzes a change I absorbance of 0,003 per minute at 253 nm at 25⁰C; pH 7.6 by the hydrolysis of BAEE = Na-benzoyl-L-arginin-rthylester).

Substrate: Na-benzoyl-L-arginine-ethylester (BAEE).

1USP unit = 1 NF according to USP XX (1980); pp 834/5: National Formulary (NF) XII pp. 646/7.

Formulation (in mg/l)

	EDTA	Trypsin	Trypsin	Trypsin/	Trypsin/
	(Versen)			EDTA-	EDTA-
					(10x)
	1%	0.25%	2.5%	0.05/0.02%	0.5/0,2%
	in PBS	in PBS	in PBS	in PBS	in
					(10x)PBS
	without	without	without	without	without
	Ca^2+Mg^2+	Ca^2+Mg^2+	Ca^2+Mg^2+	Ca^2+Mg^2+	Ca^2+Mg^2+
NaCl	8000	8000	8000	8000	80000
KCl	200	200	200	200	2000
Na2HPO4	1150	1150	1150	1150	11500
KH2PO4	200	200	200	200	2000
EDTA-Na2	10000	-	-	200	2000
Trypsin	-	2500	25000	500	5000

Trypsin/EDTA (0.05/0.02% in PBS without Ca²⁺, Mg²⁺) to passage monolayer cultures.

· Wash cell culture (monolayer) using physiological saline (w/o Ca²⁺, Mg²⁺).

· Apply a small quantity of Trypsin/EDTA with a pipette to cell culture Flask; a thin film is sufficient

· Incubate culture flask at + 37⁰C For approx. 5 mm.

· Centrifuge cell suspension or transfer directly to a serum containing culture medium. With higher trypsin concentrations, it is recommended to trypsinate at +2 - +8⁰C.

	Cat. No.	Unit
EDTA (Versen) 1%	L2113*	100ml
in PBS without Ca^2+, Mg^2+
Trypsin 2.5%	L2123	100ml
in PBS without Ca^2+, Mg^2+
Trypsin	L2133	100ml
in PBS without Ca^2+, Mg^2+
Trypsin/ EDTA(0.05%/0.02%)	L2143	100ml
in PBS without Ca^2+, Mg^2+
(10x)Trypsin/ EDTA(0.5%/0.2%)	L2153	100ml
in(10x) PBS without Ca^2+, Mg^2+
in plastic bottles
Trypsin/ EDTA(0.25%/0.02%)	L2163	100ml
in PBS without Ca^2+, Mg^2+
Trypsin powdered substance	L2103*	100g

* Storage temerature: +2 - +8 ^oC

Comparison of Collagenase and Trypsin

	Collagenase	Trypsin
1. Site of Action	collagen, reticulin and	cellular and intercellular
	other irtercellular substances	mucoproteins
2. pH optimum	6.5-7.8	7.8-8.7
3.Optimal activity	with Ca^2+ and Mg^2+	without Ca^2+ and Mg^2+
4.Impact on cells	no damage to cell membranes	damage to cell
		membranes
5.Effects of serum	collagenase may be inhibited,	clear drop in activity
	depending on serum lot and
	collagenase type, inhibition
	somewhat less as compared to
	trypsin.

PAPAIN

Storage: ≤ -20⁰C

Non-animal alternative for trypsin

The ready-to-use preparation (61.25 mg/I) in PBS w/o Ca²+/Mg²+is derived from Papaya fruit (Carica papaya). Papain is able to proteolytic decomposition (hydrolysis) of proteins and peptides, preferably those with basic amino acids.

If compared to the usual trypsin, papain preparation has a limited shelf life of 36 hrs at +2 -+8^oC Papain should consequently be thawed, divided into aliquots and re-frozen. For passage of cell cultures, supernatant is discharged and cells washed with 5 ml PBS (w/o Ca²⁺/Mg²⁺; cat. no, L1825). Supply culture flasks with (depending on size) 1.0 -2.5 ml papain. Incubate culture for 5 - 7 mm (at + 15 - +25^oC) while gently moving the flask, and observe separating progress under a microscope suitable for the survey of cells. Transfer cells into a sterile centrifugal tube and centrifuge at 300 G for 10 min. Discharge supernatant and re-suspend pellet in 15 ml culture medium. Repeat centrifugation, re-suspend pellet in medium prior to seeding it to culture flask.

	Cat. No.	Unit
Papain preparation	L2223	100 ml