||HNK-1 [HNK1, Leu7]
||Mus musculus (B cell); Mus musculus (myeloma), mouse (B cell); mouse (myeloma)
||Spleen cells were fused with P3X63Ag8.653 myeloma cells.
||immunoglobulin; monoclonal antibody; against human natural killer (NK) cells and antigen dependent killer (K) cells (CD57)
||Animals were immunized with a membrane extract of the human lymphoblastoid cell line HSB-2.
Spleen cells were fused with P3X63Ag8.653 myeloma cells.
The antibody also reacts with glycoproteins present on Schwann cells, oligodendrocytes and embryonic neurons.
The cells will not grow if the medium lacks 2-mercaptoethanol.
||RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and supplemented with 0.02 mM 2-mercaptoethanol, 80%; fetal bovine serum, 20%.
||Cultures can be maintained by addition of fresh medium.
Alternatively, cultures can be established by centrifugation with subsequent resuspension at 1 x 10e5 viable cells/mL.
Maintain cultures at a cell concentration between 1 x 10e5 and 1 x 10e6 cells/mL.
NOTE: Do not allow the cell concentration to exceed 1 x 10e6 cells/mL.
Population Doubling Time about: 24-30 hours
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells:
||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that
some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes.
4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio).
5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line).
NOTE: It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
||Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J. Immunol. 127: 1024-1029, 1981. PubMed: 6790607
Abo T, et al. Postnatal expansion of the natural killer and killer cell population in humans identified by the monoclonal HNK-1 antibody. J. Exp. Med. 155: 321-326, 1982. PubMed: 7054358
Schuller-Petrovic S, et al. A shared antigenic determinant between natural killer cells and nervous tissue. Nature 306: 179-181, 1983. PubMed: 6196639
McGarry RC, et al. Recognition of myelin-associated glycoprotein by the monoclonal antibody HNK-1. Nature 306: 376-378, 1983. PubMed: 6196641
Vincent M, Thiery JP. A cell surface marker for neural crest and placodal cells: further evolution in peripheral and central nervous system. Dev. Biol. 103: 468-481, 1984. PubMed: 6202575
McBurney MW, et al. Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture. J. Neurosci. 8: 1063-1073, 1988. PubMed: 2894413
Tucker GC, et al. Identical reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes. Cell Differ. 14: 223-230, 1984. PubMed: 6207939
||ANDREA GONÇALVES TRENTIN; UFSC- CCB