HiKaryoXLTM Nutrient Mixture F-10 Medium

With L-Glutamine, FBS, Penicillin, Streptomycin and Sodium bicarbonate Without Phytohemagglutinin (PHA-M) 1X liquid Karyotyping Medium Product Code: AL185A Product description:
HiKaryoXL Nutrient mixture F-10 Medium is a medium developed for the short term in vitro culture of peripheral blood lymphocytes for cytogenetic studies. Cytogenetic studies include metaphase and pro metaphase studies carried out on lymphocytes to detect chromosomal aberrations associated with structural and numerical abnormalities. Lymphocytes come from normal peripheral blood and are mitotically inactive, hence have to be stimulated with a mitogen. The most common mitogen used for the stimulation of cell division in lymphocyte cultures is Phytohemagglutinin (PHA-M). Phytohemagglutinin is a lectin extract from red kidney bean (Phaseolus vulgaris). The protein consists of two subunits, a leucoagglutinin (PHA-L) and an erythroagglutinin (PHA-E). PHA-M is the mucoprotein form and is used for stimulation of cell proliferation in lymphocyte culture. HiKaryoXL Nutrient Mixture F-10 Medium is a karyotyping medium composed of a basal medium Nutrient Mixture F-10 and supplemented with L-Glutamine, FBS, Penicillin, Streptomycin and Sodium bicarbonate. It does not contain PHA-M, hence if required, has to be added prior too use. L-Alanine L-Arginine hydrochloride L-Asparagine anhydrous L-Aspartic acid L-Cystine dihydrochloride L-Glutamic acid L-Glutamine L-Histidine hydrochloride monohydrate L-Isoleucine L-Leucine L-Lysine hydrochloride L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine disodium salt L-Valine
VITAMINS

8.910 211.000 15.010 13.300 35.130 14.700 146.000 21.000 2.600 13.100 29.300 4.480 4.960 11.500 10.500 3.570 0.600 2.610 3.500 0.698 0.024 0.715 1.320 0.615 0.206 0.376 1.000 1.360 0.541 1100.000 Proprietary 4.080 0.210 Proprietary 1.300 Proprietary 110.000 0.730

Composition:
Ingredients
INORGANIC SALTS

mg/L 44.100 0.0025 0.834 74.730 285.000 83.000 1200.000 7400.000 153.700 0.0288 7.510

Calcium chloride dihydrate Copper sulphate pentahydrate Ferric sulphate heptahydrate Magnesium sulphate anhydrous Potassium chloride Potassium dihydrogen phosphate Sodium bicarbonate Sodium chloride Sodium phosphate dibasic anhydrous Zinc sulphate heptahydrate
AMINO ACIDS

Choline chloride D-Biotin D-Ca-Pantothenate Folic acid Niacinamide Pyridoxine hydrochloride Riboflavin Thiamine hydrochloride Vitamin B12 i-Inositol
OTHERS

Glycine

D-Glucose Fetal Bovine Serum Hypoxanthine sodium salt Lipoic acid Penicillin Phenol red sodium salt Streptomycin Sodium pyruvate Thymidine

Directions:
1. 2. Add PHA-M to HiKaryoXL Medium in required concentration (5-15g/ml) prior to inoculation of blood. Add whole blood to 10ml of this medium containing PHA-M in T-25cm2 flasks as per the following recommendations: Normal adults - 0.8ml Infants and children- 0.6ml Women (during pregnancy/ postpartum) - 1.0ml Note: If required,. Incubate the flasks at 37OC and 5% CO2 for 67-72 hours. To determine optimum incubation time i.e. the peak mitotic index, collect samples at different time intervals between 48-72 hours. Note: Peak mitotic index is most commonly observed at 67-72 hrs. Add 100l of 10g/ml of colchicine and incubate for additional 2 hours. Note: Incubation time of less than 1 hour might result in reduced mitotic index Transfer the entire content of the flask to a sterile centrifuge tube and centrifuge at 800-1000rpm for 10 minutes. Discard the supernatant and resuspend the pellet in 5ml of hypotonic 0.075M KCl solution and incubate in a water bath at 37oC for 15-20 minutes. Note: Add KCl solution drop wise while agitating the cells Add equal amount of freshly prepared ice cold fixative (Acetic acid : methanol, 1:3 parts) Centrifuge cells at 800-1000rpm for 10min. Discard the supernatant and again add 5ml of freshly prepared ice-cold fixative (acetic acid: methanol, 1:3 parts) with constant mixing. Leave the cells at 4oC for 10-15 min. Centrifuge the cells at 1000rpm for 10 minutes. Repeat step no. 10 and 11. Discard the supernatant and resuspend the pellet in 0.2ml of fresh fixative. Put 1 drop of cell suspension on to a clean, cold slide. Tilt the slide and let the drop run down the slide as it spreads and dry the slide rapidly over a hot plate or a beaker of boiling water. Stain the slides as required.
TM

Quality control:
Appearance Orangish colored, clear solution pH 7.00 -7.60 Osmolality in mOsm/Kg H2O 320.00 -360.00 Sterility No bacterial or fungal growth is observed after 14 days of incubation, as per USP specification. Cultural Response The growth promotion capacity of the medium is assessed qualitatively by analyzing the cells for the morphology and quantitatively by estimating mitotic index.

3. 4.

5.

Storage and shelf life:

Store at -20C. The medium can be aseptically transferred into smaller aliquots for convenience. Once thawed, use the medium within 30 days. Avoid exposure to light. Shelf life is 12 months. Use before expiry date given on the product label.

6.

7.

Following troubleshooting tips can be used in case of no growth or slow growth:

Cause Toxic collection equipment Specimen received in lithium heparin, EDTA, phenol-preserved heparin, or other wrong preservative Improper shipment or storage of sample Solution Check collection equipment. Do a controlled experiment to rule out toxicity. Request another specimen if possible. Wash blood several times before setting up to increase chances of growth. Check shipment variables: time passed between collection and receipt, temperature control, pressure control, proper pH of transport medium.

8. 9. 10.

11. 12. 13. 14.

15.

Materials required but not provided:

HiKaryoXL PHA-M solution 0.1mg/ml (TCL061) HiKaryoXL PHA-M solution 1mg/ml (TCL071) HiKaryoXL Colchicine Solution (TCL062) HiKaryoXL Colcemid Solution (TCL074) Potassium Chloride solution 0.075M (TCL040) Methanol Acetic Acid, Giemsa Stain (TCL083)

or

Faulty glassware or plastic Try another lot or brand of ware pipette, flask, Petri dish, coverglass etc. PHA- unresponsive culture Try pokeweed mitogen, Concanavalin A, another mitogen, or two mitogens. Repeat the test at a later date if possible.
Revision: 1/ 2010-06

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