M1/70.15.11.5.HL

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

HANDLING PROCEDURE FOR FROZEN CELLS

- Initiate culture as soon as possible upon receipt.

- Thaw by rapid agitation in 37øC water bath. Thawing should be rapid (within

40-60 seconds). As soon as the ice is melted, remove the ampule from the water

bath and immerse in 70% ethanol at room temperature. All of the operations from

this point on should be carried out under strict aseptic conditions.

- The cells are supplied in two different types of glass ampules. One is a

standard ampule, the neck of which must be scored with a sharp file that has

been immersed in ethanol. A definitive sharp nick about 1/8" in length on one

side is necessary. The second type is prescored and is identifiable by a gold

band around the ampule neck, and should not be scored with a file.

- Break the neck of the ampule between several folds of a sterile towel.

- Transfer the cell suspension and dilute it with the recommended culture

medium in a culture flask (see specific batch information above for dilution

ratio); incubate at 37øC with 10% CO2 in air atmosphere. Since it is important

to avoid excessive alkalinity of the medium during recovery of the cells, it is

suggested that the culture medium be placed into the culture flask, tube, etc.

and the pH be adjusted, as necessary, prior to the addition of the ampule

contents. Note that the bicarbonate content of the culture medium will

determine whether an atmosphere containing CO2 will be required.

- It is not necessary to remove the freezing additive. However, if desired, the

culture medium may be changed to remove the protective freezing additive

(dimethylsulfoxide) 24 hours after thawing. If it is desired that the freezing

additive be removed immediately, or that a more concentrated cell suspension be

obtained, centrifuge the above diluted suspension at approximately 125 x g for

10 minutes, discard the fluid and resuspend the cells with growth medium at the

dilution ratio given in the specific batch information above.

FLUID RENEWAL

Add fresh medium (depending on cell density) every 2-3 days.

SUBCULTURE PROCEDURE

Cultures can be maintained by the addition of fresh medium or the replacement

of medium. Adherent cells can be dislodged by scraping and cultures established

by centrifugation with subsequent resuspension at 1-2 x 10(5) viable cells/ml.

Maintain cell culture density between 10(5) and 10(6) viable cells/ml.

_______________________________________________________________________________

HANDLING PROCEDURE FOR FLASK CULTURES (SUSPENSION)

The flask was seeded with cells (see specific batch information above for

concentration), grown and completely filled with medium to prevent loss of

cells in transit. Upon receipt incubate the flask in an upright position for

several hours to return the flask contents to 37øC. After the temperature has

equilibrated, aseptically remove the entire contents of the flask and

centrifuge at 300 x g for 15 minutes. Resuspend the cell pellet in 10-12 ml of

the shipping medium. From this suspension remove a sample for a cell count and

viability so that the cell density of the suspension can be adjusted to 1-2 x

10(5) viable cells/ml. If the suspension needs to be diluted use the shipping

medium. Incubate the culture in a flat position at 37øC. The shipping medium

contains reduced sodium bicarbonate suitable for a 5% CO2 in air incubator.

DMEM usually contains 3.7 grams of sodium bicarbonate per liter and should be

incubated in a 10% CO2 in air incubator. Maintain the cell density of the

culture as suggested under the subculture procedure described above.

_______________________________________________________________________________

NOTE

This material is available under the conditions that you will not use it for

commercial purposes or distribute it to third parties.

_______________________________________________________________________________

CATALOGUE DESCRIPTION

This hybridoma secretes a monoclonal IgG2b antibody which reacts with the alpha

chain of the murine macrophage-granulocyte specific antigen Mac-1 (CD116).

Mac-1 is a macrophage differentiation antigen associated with type three

complement receptor. The antigen defined by this hybridoma is expressed in

large quantities on thioglycollate peritoneal exudate macrophages and in lesser

amounts on neutrophilic granulocytes, blood monocytes, 8% of spleen cells, 44%

of bone marrow cells, and less than 0.3% of thymus cells. This antibody

precipitates 2 polypeptides of 190,000 and 105,000 daltons. This antibody

labels human monocytes and polymorphonuclear leukocytes and a small population

of lymphocytes. It is capable of both natural killing (NK) and

antibody-dependent cellular cytotoxicity (ADCC). The hybridoma was formed by

the fusion of mouse myeloma line NS-1 with spleen cells from DA rats immunized

with C57BL/10 mouse spleen cells enriched for T lymphocytes. References: Eur.

J. Immunol. 8: 539-551, 1978; ibid., 9: 301-306, 1979; J. Biol. Chem. 256:

3833-3839, 1981; Monoclonal Antibodies, R. Kennett, et al. (eds.), pp. 185-217,

Plenum Press, 1980; J. Exp. Med. 158: 586, 1983. Originator: T. Springer,

Sidney Farber Cancer Institute, Harvard Medical School, Boston, MA.

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Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.