SEPARATING SOLUTIONS AND LECTINS

| Biocoll | Percoll | Mitogens |

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BICOLL CELL separating soLUTION

Storage +2 - +25°C

Biocoll separating solution does contain a polymer with a molecular weight of approx. 400.000 Dalton. Densities of up to 1.1 g/ml can be adjusted using this hydrophilic polymer. For optimal pH and osmolality, adjusting Biocoll with an acid, preferably amidotrizoeic acid (ATA), and sodium hydroxide is required. Biocoll with densities of 1.077 g/ml (cat. no. L6113/5) and 1.090 g/ml (cat. no. L6125) are already adjusted accordingly.

Instruction how to dilute the stock solution of Biocoll (density 1.090 g/ml; cat. no.L6125) with PBS (cat. no. L1823/0):

desired	vol % of	PBS
density (g/ml)	Biocoll 1.090	(vol %)
1.080	88.2	11.8
1.075	82.4	17.6
1.070	76.5	23.5
1.065	70.6	29.4
1.060	64.7	35.3
1.055	58.8	41.2
1.050	52.9	47.1
1.045	47.1	52.9
1.040	41.2	58.8
1.035	35.3	64.7
1.030	29.4	70.6

All amounts given for a temperature of +20°C. Only PBS w/o Ca²⁺/Mg²⁺ is suitable, since these ions influence agglomeration and increase formation of cell clusters.

separating soLUTION - percoll®

Storage +2 - +8^oC

The basis of Percoll are silica gel particles, coated with PVP (polyvinyl pyrrolidone). BIOCHROM ready-to-use preparations are already adjusted to physiological values of osmolality and pH. Any desired densities from Percoll (cat. no. L6143/5) can conveniently be prepared with PBS (cat. no. L1823/5), using the formula:

Vol. % Percoll (1.124 g/ml) =

desired	Percoll	PBS
density in g/ml	Cat.No.	Cat.No.
	L6143 / 5	L1825
1.063	48.74 ml	51.26 ml
1.068	52.94 ml	47.06 ml
1.077	60.50 ml	39.50 ml
1.100	79.83 ml	20.17 ml
1.113	90.76 ml	9.24 ml

All amounts are given for a temperature of +20°C. Only PBS w/o Ca²⁺/Mg²⁺ is suitable, since these ions might influence agglomeration and hence separating of cell clusters.

Biocoll and Percoll separating solutions

Parameter	Biocoll+ Amidotrizoic acid-Na-salt	Percoll + PBS
Viscosity	3.6 cP at +20^oC	1.5 cP at +20^0C
		more preserving separation,
		better yield.
Cytoxicity	inhibition of mitosis following long contact,	no inhibition
	alteration of cell density.	no density alterations
Phagocytosis	none	monocytes may ingest silicon particles,
		but are not activated.

Storage +2 - + 25^oC

	Cat. No.	Unit
Biocoll separating solution	L6113	100 ml
Density 1.077 g/ml, isoton	L6115	500 ml
Biocoll separating solution	L6125	500 ml
Density 1.09 g/ml, isoton
Biocoll separating solution	L6155	500 ml
Density 1.10 g/ml, isoton

Storage +2 -+8°C

	Cat. No.	Unit
Percoll separating solution	L6133	100 ml
Density 1.077 g/ml, isoton	L6135	500 ml
Percoll separating solution	L6143	100 ml
Density 1.124 g / ml, isoton	L6145	500 ml

PERCOLL 40 / 80 %

Storage+2-+8°C

Percoll 80%, and Percoll 40% are diluted using Ham's F-10 (10x) in a ratio of 8:2 (Percoll 80), and 4:6 (Percoll 40).Osmolality is adjusted to 275 - 297 mosm/kg H₂O, and pH to 7.24 ± 0.25.

	Cat. No.	Unit
Percoll 80%	L6173	100 ml
Density 1.102 g/ml
Percoll 40 %	L6163	100 ml
Density 1.055 g/ml

Separating lymphocytes using Biocoll

· Biocoll separating solution (D=1.077at+20°C) is given to 15 or 25 ml-centrifugal tubes In volumes of 7 ml and 10 ml, resp.

· Equal parts of heparinized whole blood (50 l U/ml heparin, stabilisator free) and culture medium are mixed and carefully applied over Biocoll separating solution.

· Centrifuge 1200 G for 20 min.

· Take layer of enriched (70 -100%) lymphocytes (between plasma and Biocoll) with a Pasteur's pipette and wash twice in culture medium:

1. for 10 min at 300 G

2. for 10 min at 200 G

· Cell counting as usual.

Separating mononuclear cells (lymphocytes and monocytes) from whole human blood using Percoll

· Put 6 ml of Percoll (1.077 g/ml) info a centrifugal tube of 25 ml.

· Dilute heparinized whole blood (25 U/ml) by an equal volume of culture medium or physiological saline.

· Carefully place diluted whole blood on Percoll (up to 20 ml per tube) and centrifuge at 400 G for 30 min at room temperature.

· Mononuclear cells are readily visible as a white stratum between plasma and Percoll, erythrocytes together with granulocytes are found in the sediment.

· Mononuclear cells are harve-5ted with a Pasteur's pipette.

· Cell suspension is diluted with an equal volume of culture medium or saline, well mixed and washed 2 or 3 times in culture medium (250 G/10 min at+2 - +8°C).

Media - suitable for lymphocyte culture

	Cat. No.
RPMI 1640 - liquid medium	F1213 / F1215
RPMI 1640- liquid medium	F1235
Medium 199 with Earle's salts - liquid medium	F0663 / F0665
RPMI 1640 with stable glutamine - liquid medium	FG1383 / FG1385
VLE RPMI 1640	F1415
VLE RPMI 1640 with stable glutamine	FG1415

MITOGENS (LECTINS)

Storage +2 - +8°C

About 100 years ago, a pro-tein, extracted from Canavalia ensiformis, was found to agglutinate human erythrocytes- The surified protein was named Concanavalin A (Con A). Similiar agglutinins from other plant species were identified (80 % of them are leguminose species), that agglutinate erythrocytes either specific or blood group specie.They were named as phytohemagglutinins.

Molecular basis of agglutination:

Phytohemagglutimns do have several binding sites for carbohydrate derivates located in the cell membrane. For instance, Con A readily binds to mannose and glucose. PHA recognizes more complex oligosaccharides. By means or multiple binding, next-neighbour cells are agglutinated.

Molecular basis of mitogenic stimulation:

Lymphocytes proved to have an increased mitosis rate under the influence of this phytohemagglutinin. Binding of the mitogen to the cell membrane seems to increase the molecular/information flow between the cell and its environment. Increased membrane permeability activates the synthesis of proteins and nucleic acids, The synthesis rare for messenger substances like lymphokines (e.g. IL-2) is also increased.

Nomenclature

Earlier nomenclature f phytohemagglutinies mostly referred to their purification level: PHA M, e.g., was named after residual content of mucoprotein, whereas PHA P indicated the pure protein.

The recent nomenclature, however, rather refers to biological characteristics: PHA P preferrably agglutinizes erytrocytes, and is therefore now termed PHA E (cat.no. M 5040). PHA M (cat.no. M 5030) was similarly re-named to PHA L, due to its particularly stimulating effect on lymphocytes.

	Cat. No.	Unit	Protein Content
Phytohemaaglutinin (PHA) L	M5030	1 x (6 x 5 ml)	1.2 mg
- lyophilized, for 5 ml
Phytohemaaglutinin (PHA) E	M5040	1 x (6 x 5 ml)	2.4 mg
- lyophilized, for 5 ml
Concanavalin A (Con A)	M5050	1 x (6 x 5 ml)	0.4 mg
- lyophilized, for 5 ml
Pokeweed Mitogen (PWM)	M5060	1 x (6 x 5 ml)	0.4 mg

Following re-constitution in 5 ml A. dest., apply 1 ml per 100 ml medium.

Specific activity of these lectins is routinely tested in a short time assay (72 – 80 hrs) using peripheric human lymphocytes. The following test protocol applies:

· Separation of human lymphocytes by means of Biocoll density centrifugation

· 1 x 10 lymphocytes/ml (0,2 ml in microplates)

· Culture medium: RPMI 1640, 20 mM HEPES, 10 % pre-tested FBS

· 10 µ/ml lectin

· Radioactive labelling: 0,2 µCi 3H-thymidine per test plate (spec. act. 2Ci/mM)

· Culture: 72 – 80 hrs at 37⁰C

Lectin content is then standardized according to maximum stimulation values.