Peripheral Blood Karyotyping Solutions

Human Cytogenetics

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HiKaryoXL™ Karyotyping Media

HiKaryoXL™ media are a series of media developed for short term in vitro culture of peripheral blood lymphocytes for cytogenetic studies. These media are based on either RPMI 1640 or Nutrient Mixture F-10 Ham and are further supplemented with L-Glutamine, FBS, Penicillin, Streptomycin and Sodium bicarbonate. They are supplied with or without PHA-M to suit the convenience of users.

Product Name Code Packing

HiKaryoXL™ RPMI Medium With L-Glutamine, FBS, PHA-M, Penicillin, Streptomycin and Sodium bicarbonate

AL165A-50X10ML AL165A-5X100ML

50x10ml 5x100ml

HiKaryoXL™ RPMI Medium With L-Glutamine, FBS, Penicillin, Streptomycin and Sodium bicarbonate Without PHA-M


50x10ml 5x100ml

HiKaryoXL™ Nutrient Mixture F10 Medium With L-Glutamine, FBS, PHA-M, Penicillin, Streptomycin and Sodium bicarbonate


50x10ml 5x100ml

HiKaryoXL™ Nutrient Mixture F10 Medium With L-Glutamine, FBS, Penicillin, Streptomycin and Sodium bicarbonate Without PHA-M


50x10ml 5x100ml

Advantages

Excellent for short term culture of peripheral blood lymphocytes

Stimulates the proliferation of peripheral blood lymphocytes

No addition of serum, L-Glutamine or antibiotics required

Ready- to-use after thawing

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Directions for use

1. Add freshly collected heparinised whole blood to 10ml of HiKaryoXLTM Medium in two T-25cm2 non-treated �asks as per the following recommendations i. Normal adults - 0.8ml ii. Infants and children - 0.6ml iii. Women (during pregnancy/ postpartum) - 1.0ml

2. Incubate the �asks at 37°C and 5% CO2 for 70-72 hours in upright position.

3. To determine optimum incubation time i.e. the peak mitotic index, collect samples at di�erent time intervals between 48-72 hours. Note: Peak mitotic index is most commonly observed at 70-72 hrs.

4. Add 100µl of 10µg/ml of colchicine (TCL062) and incubate for additional 2 hours. Note: Incubation time of less than 1 hour might result in reduced mitotic index.

5. Transfer entire content of the �ask to a sterile centrifuge tube and centrifuge at 800-1000rpm for 10 minutes.

6. Discard the supernatant and resuspend the pellet in 5ml of warm 0.075M KCl hypotonic solution (TCL040) and incubate in a water bath at 37°C for 15-20 minutes. Note: Add KCl solution drop wise while agitating the cells.

7. Add 5ml of freshly prepared ice cold �xative. (Acetic acid: methanol, 1:3 parts)

8. Centrifuge cells at 800-1000rpm for 10min.

9. Discard the supernatant and again add 5ml of freshly prepared ice-cold �xative (acetic acid: methanol, 1:3 parts) with constant mixing. Leave the cells at 4°C for10-15 min.

10. Centrifuge the cells at 1000rpm for 10 minutes.

11. Repeat step no. 9 and 10.

12. Discard the supernatant and resuspend the pellet in 0.2ml of fresh �xative.

13. 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 air dry the slide.

14. Fix the smear over a hot plate or beaker of boiling water and stain the slides as required.

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HiKaryoXL™ Karyotyping Reagents

Mitotic StimulatorsLymphocytes from peripheral blood are mitotically inactive and have to be stimulated with a mitogen. In presence of a mitogen, small lymphocytes undergo a process known as transformation, in which the cell enlarges and the staining properties of the nucleus change. Such a transformed cell is capable of cell division.

Lymphocytes in puri�ed preparation or in whole blood can be stimulated with di�erent mitogens such as Phytohemagglutinin (PHA), Concanavalin A, Pokeweed mitogen (PWM) etc. Phytohemagglutinin (PHA) and Concanavalin A a�ect primarily the T cell population while Pokeweed mitogen (PWM) a�ects the B cell population.

HiKaryoXL™ PHA-M SolutionThe most common mitogen used for the stimulation of cell division in lymphocyte cultures is Phytohemagglutinin (PHA). PHA causes small T lymphocytes to transform to lymphoblasts and enter mitosis. PHA acts by causing a marked increase in RNA synthesis within the �rst 24 hours. Lymphocytes produce interleukin-2 (IL-2) or lymphocyte growth factor which further stimulates mitosis. DNA synthesis is low during the �rst 30 hours of culture but it increases steadily between 30 and 60 hours.

The use of PHA-M as a mitogen helps to obtain lymphocyte that are actively dividing, thus yielding analyzable mitotic chromosome spreads. PHA-M is a lectin extracted from red kidney bean Phaseolus vulgaris. The protein consists of two subunits, a leucoagglutininin (PHA-L) and an erythroagglutinin (PHA-E). PHA-M is the mucoprotein form and is most commonly used in cytogenetics laboratories.


HiKaryoXL™ PHA-M solution With 0.1mg per ml PHA-M in sterile tissue culture grade water

TCL061-1X10ML 1x10ml

HiKaryoXL™ PHA-M solution With 1mg per ml PHA-M in sterile tissue culture grade water


PHA-M Cell Culture Tested

TC209-10MG TC209-25MG TC209-4X25MG

10mg 25mg 4x25mg

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PHA-PPhytohemagglutinin (PHA-P) is a lectin isolated from red kidney beans Phaseolus vulgaris. It is puri�ed by a�nity chromatography. PHA-P has a molecular weight of 115kDa. The lectin PHA-P consists of �ve glycoproteins that are tetrameric structures made up of two subunits PHA-E (erythroagglutinin) and PHA-L (leucoagglutinin). PHA-E has a low mitogenic activity and a high erythroagglutination activity whereas PHA-L has a high mitogenic and leucoagglutinating activity, but very low erythroagglutinating activity. PHA-E is not blood group speci�c but agglutination can be inhibited by certain oligosaccharides.


PHA-P Cell Culture Tested

TC226-5MG TC226-5X5MG

5mg 5x5mg

Concanavalin AConcanavalin A (Con A) is a glycoprotein isolated from Jack bean (Canavalia ensiformis). Con A speci�cally binds with speci�c terminal sugar residues like α-mannose and α-galactose structures found in sugars, glycoproteins and glycolipids. It agglutinates red blood cells and complexes with blood group substances, immunoglobulin, glycopeptides and carcinoembryonic antigens. Hence it is widely used in hormone receptor studies, mitogenic assays and for characterizing normal and malignant cells. It is also used to initiate mitogenesis in T lymphocytes by stimulating the energy metabolism of thymocytes.

In neutral and alkaline solutions, concanavalin A exists as a tetramer consisting of 4 subunits of 26.5kDa each. In acidic solutions (pH below 5.0), concanavalin A exists as a dimer.


Concanavalin A Cell Culture Tested

TC220-25MG TC220-100MG

25mg 100mg

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Mitotic InhibitorsProper techniques for harvesting dividing cells and preparing slides for chromosomal analysis are critical for attaining the quality needed for correct karyotype analysis. Chromosomes become structurally and numerically distinct only during the metaphase stage of cell division. Mitotic inhibitors like Colchicine and Colcemid are used to collect the cells at this stage for cytogenetic analysis. Mitotic inhibitors disrupt mitotic spindle �bers and free the chromosomes from metaphase plate, accounting them to spread out inside the cell. The lack of spindle �bers also blocks the anaphase so the mitotic index is e�ectively increased. Mitotic inhibitors also cause chromosome contraction. The degree to which chromosomes contract depends on the concentration and also the time that the cells are exposed to the mitotic inhibitors.

HiKaryoXL™ Colchicine SolutionColchicine, an alkaloid isolated from the plant Colchicum autumnale, is a microtubule-depolymerizing agent that has been used to arrest cells at metaphase. Arresting of cells in metaphase allows an increased yield of mitotic cells for analysis. Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules. Availability of tubulin is essential to mitosis, and therefore colchicine e�ectively functions as a “mitotic poison” or spindle poison.


HiKaryoXL™ Colchicine solution With 10µg per ml Colchicine in Phosphate bu�ered saline


HiKaryoXL™ Colcemid® SolutionColcemid® also known as demecolcine, is related to colchicine but it is less toxic. It depolymerises microtubules and limits microtubule formation (inactivates spindle �bre formation), thus arresting cells in metaphase and allowing cell harvest and karyotyping to be performed.


HiKaryoXL™ Colcemid® solution With 10µg per ml Colcemid in Hank’s balanced salt solution

TCL074-1X20ML TCL074-1X100ML

1x20ml 1x100ml

Colcemid® is a registered trademark of Ciba - Giegy Corp.

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Stains

Giemsa StainG-banding is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes. Banding can be used to identify chromosomal abnormalities because there is a unique pattern of light and dark bands for each chromosome. The metaphase chromosomes are treated with trypsin and stained with Giemsa.

Product Name Code PackingGiemsa Stain Solution TCL083-1X100ML

TCL083-1X500ML1x100ml 1x500ml

Giemsa Stain Cell Culture Tested

TC232-5G TC232-25G

5gm 25gm

Hoechst 33258 & Hoechst 33342Hoechst, a bis-benzimidazole derivative compound, is a DNA intercalator. It preferentially binds to adenine-thymine (A-T) regions of DNA. It is excited by ultraviolet light at around 350 nm and emit blue/cyan �uorescence around an emission maximum at 461 nm. Hoechst stain may be used on live or �xed cells. It is also used to stain the metaphase chromosomes with much brighter �uorescence and di�erent banding pattern. Hoechst stain shows minimal background �uorescence and slow quenching. Ability of Hoechst 33342 to permeate the cells is about 10 times higher than Hoechst 33258.

Product Name Code PackingBisbenzimide (Hoechst 33258) Cell Culture Tested

TC225-25MG TC225-100MG

25mg 100mg

Bisbenzimide (Hoechst 33342) Cell Culture Tested

TC226-25MG TC226-100MG

25mg 100mg

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Related Reagents

Potassium Chloride Solution, 0.075MA hypotonic solution of potassium chloride is used in blood lymphocyte chromosome preparation. The hypotonic treatment causes the cells to swell and aids in the release of the intact chromosomes. The KCl hypotonic treatment is important for swelling of the cells and adequate spreading of chromosomes on the slide.


Potassium Chloride solution, 0.075M TCL040-1X100ML 1x100ml

Trypsin-EDTA SolutionsG-banding requires pretreatment of chromosomes with trypsin to partially digest the chromosomes which are then stained with Giemsa stain. Each homologous chromosome pair has a unique pattern of G-bands, enabling recognition of particular chromosomes.


Trypsin-EDTA Solution 10X With 2.5% trypsin (1:250), 0.2% EDTA in 0.85% normal saline

TCL070-1X100ML TCL070-5X100ML TCL070-2X500ML

1x100ml 5x100ml 2x500ml

Trypsin Solution for G-Banding w/ 0.025% Trypsin in Dulbecco’s Phosphate Bu�ered Saline


DAPI- 4’, 6-Diamidino-2-phenylindoleDAPI (diamidino-2-phenylindole) is a �uorescent stain that binds strongly to the DNA. It is rapidly taken up by the cellular DNA because of its high cell permeability. It selectively binds to the minor groove of double stranded DNA. The excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm. DAPI can be used for both �xed and live cell staining, though the concentration of DAPI needed for live cell staining is generally much higher than for �xed cells.

Product Name Code PackingDAPI (4’,6-Diamidino-2-phenylindole) Cell Culture Tested

TC229-5MG TC229-10MG

5mg 10mg

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Gurr Bu�er SolutionGurr bu�er is a used for G-banding of chromosomes by Giemsa staining for cytogenetic analysis.


Gurr Bu�er Solution pH 6.8 TL1139-1X500ML 1x500ml

HiPer® G-Banding Teaching Kit

G-banding is the most widely used banding method for chromosome analysis. It is also known as GTG banding (G bands produced with Trypsin and Giemsa). Prepared and aged slides are treated with the enzyme Trypsin and then stained with Giemsa. This produces a series of light and dark bands that allow positive identi�cation of each chromosome. The dark bands are A-T rich, late replicating, heterochromatic regions of the chromosomes while the light bands are C-G rich, early replicating, euchromatic regions. The light bands are biologically more signi�cant because they represent the active regions of the chromosomes while the dark bands contain relatively few active genes. In addition to a distinct banding pattern, individual chromosomes are identi�ed according to their size and centromere position.


HiPer™ G-Banding Teaching Kit CCK043-1KT 1 Kit

Kit Contents

Trypsin Solution for G BandingGiemsa Stain Solution 20XFetal Bovine SerumGurr Buffer SolutionDemonstration Slide

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Troubleshooting Tips

Problem Cause Solution

No cell growth or very slow growth

Blood used for culture is not fresh

Always use freshly drawn blood

CO2 percentage in the incubator too high or too low

Check percentage of CO2 inside the incubator. It should be 5 ± 0.5%

Incubation temperature too high or too low

Check incubator temperature. It should be 37°C ± 0.5°C. Lower temperatures retard the growth rate. Higher temperatures usually result in cell death.

No chromosomes or scattered chromosomes

Cells burst during harvest procedure

Ensure gentle addition of fixative and hypotonic solution

No metaphases Harvesting not performed in exponential phase

Harvesting should be done between 70 - 72 hours

Chromosomes not well spread or non-uniform

Presence of cell aggregates Disperse cell clumps before dropping the cell suspension on slide

Drop the cell suspension on the slide from a height

Non uniform drying of slide Avoid blowing and always air dry the slide

Debris on slide affecting drying in some spots

Ensure that the slides are clean and grease-free

Contracted Chromosomes

Prolonged treatment with mitotic inhibitor

Repeat the procedure by treating the culture with mitotic inhibitor for recommended time

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Peripheral Blood Karyotyping Solutions

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