The peripheral blood lymphocyte culture is one of the most routinely used and traditional methods for karyotyping analysis.

T- lymphocyte elements of the whole blood are used for Peripheral Blood Lymphocyte culture. 

The idea behind the cell culture is to obtain a metaphase chromosome. The metaphase chromosomes are a well-separated form of chromosome having 2n numbers of chromosomes in it. 

In the present article, we will give you our standard PBLC protocol you can use it in your own lab. 

Read our previous article of this series: A Brief Introduction To Cytogenetics [Karyotyping, FISH and Microarray]

Requirements: 

Utilities:  

15ml falcon tubes, heparin tubes, culture flask, micropipette, pasture pipette, 1 to 10ml pipette, beaker, mouth cap, head cap, gloves and tips. 

Chemicals: 

Alcohol, methanol, Glacial acetic acid, RPMI-1640, Streptomycin, penicillin, L-glutamine, EtBr, colchicine or colcemid, KCl, Giemsa, trypsin and water. 

Instruments:  

Laminar airflow or class III safely cabinet, centrifuge, microscope, incubator and refrigerator. 

Chemical preparation:

100mg/ml Penicillin:

100mg of penicillin dissolve in 1000µl of sterile distilled water, store in a cool place. 

100mg/ml streptomycin:

Dissolve 100mg of streptomycin in 1000µl of sterile distilled water, store in the cool place. 

200mM L-glutamine: 

Dissolve 0.029gm of L-glutamine in 1000µl of sterile distilled water, prepare freshly just before use. 

1mg/ml EtBr: 

Dissolve 1mg of EtBr in 1000µl of sterile distilled water, store in a cool place. 

2mg/10ml of colchicine: 

Dissolve 2mg of colchicine in 10ml of sterile distilled water, store in a cool place. 

Hypotonic solution (0.56% KCl): 

Dissolve 0.56 gm of KCl in the 100ml of sterile distilled water, prepare it just before the use (pH 7.2 to 7.4). 

Fixative: 

Mix three-part of methanol and 1 part of glacial acetic acid, prepare it at the time of use, and chilled it properly at -20℃. 

Giemsa stain solution: 

The 1gm of Giemsa dissolved in 66ml of glycerol using the mortar and pestle for 45 minutes. 

Transfer it into the aluminum foil wrapped flask and heat steer for 45 minutes to 1 hour. 

Place it at room temperature for some time and add 84ml of methanol to it. 

Again steer it for 1 hour. 

Place for room temperature for some time and transfer it to the dark amber bottle. 

Our stock solution of Giemsa it ready. 

Before use, mix 5ml of stock Giemsa solution with 45ml of distilled water. 

Note: all the chemicals are available in the ready to use form, you can also purchase it to avoid all these preparation steps. 

Role of each chemical in PBLC:

RPMI 1640:  

The RPMI 1640 is a modification of McCoy’s 5A medium which is specially prepared for the PBLC. 

It is a growth medium for the tissue culture which provides all the essential ingredients such as vitamins, inorganic salts and amino acids necessary for the growth of cells. 

L-Glutamine:  

The L-glutamine is an essential amino acid required for the cell growth of all the mammalian and insect cells. 

It is used separately because it is unstable, therefore, it must be prepared freshly all the time before cell culture. 

Antibiotics:  

Bacterial fungal contamination is most commonly observed in the cytogenetic tissue culture. Use of antibiotics prevents the contamination of growing cell lines. 

In the peripheral blood lymphocyte culture, penicillin and streptomycin are two antibiotics regularly used. 

Role of colchicine/colcemid:

 Using the colcemid in the tissue culture prevents spindle fiber formation during the cell division. This action will arrest the cell in the metaphase stage, consequently, the cell cannot be entered in the anaphase. 

Role of EtBr:  

It is believed that adding EtBr before the cell harvesting will release the condensation of the chromosome and loosen the compactly packed chromosome. 

Due to this, the chromosome appears more elongated and larger. 

Role of the hypotonic solution:  

The hypotonic solution allows the intake of water that results in the cell swelling and cell burst. 

This can helps in the separation of chromosomes. 

Role of fixative:

 The fixative will help in washing the cells, it removes other cell debris. 

Phytohemagglutinin (PHA)-P:  

The PHA-P is a mitotic stimulator that promotes the cell division during the tissue culture. It is provided in the culture medium. 

Principle of PBLC: 

The T- lymphocyte cells cannot undergo cell division further, adding the mitotic agent PHA-P (already present in the RPMI 1640 medium) stimulate the cell division in the cell culture. All the cells are arrested at the metaphase by adding the colcemid or colchicine. 

Treating cells with the EtBr, hypotonic solution and fixative will promote chromosomal elongation, cell swelling & bursting and separation, respectively. 

By staining with the Giemsa solution the chromosome can be microscopically visible.   

Read our article on chromosome: Story of Chromosome

Karyotyping protocol for Peripheral Blood Lymphocyte Culture:

The Peripheral blood lymphocyte culture method is divided into the following steps: 

Sample collection: 

2 ml of the whole blood sample is collected in the heparin tube, strict aseptic conditions are maintained during the sample collection. 

The sample must proceed under 24 hours. However, before doing the culturing, place sample for some time at room temperature. 

Cell culture: 

One of the most important procedures in the cytogenetic analysis is to maintain aseptic conditions, therefore, before doing the cell culture check all the instruments and reagents used for the culturing whether it is contamination free or not. 

Wipe the laminar airflow with the alcohol. Wear gloves, mask and head cap. Further, wear a clean lab coat. 

Do not open any of the reagents until now. 

Now clean all the plasticware and glassware with alcohol. Clean your hands with alcohol too. 

Now add 7ml of RPMI-1640 compete-media to the 15 ml falcon tube, Carefully close the bottle and cover it with parafilm. 

 Add 100mg/ml 10μl penicillin and 20μl streptomycin antibiotics to the culture tube. 

Add 200mM of 100µL L-glutamine followed by the addition of 100µL of phytohemagglutinin M. 

Now carefully add 0.7ml of a blood sample in the culture tube. 

The culture is mixed well by inverting gently several times and incubated at 37℃ in the incubator for 70 to 72 hours. 

Remember, the tubes are placed at the angle of 45 degrees, therefore, the culture can grow efficiently.  

Note: The incubator used for the PBLC must be clean and contamination-free also it is very necessary that this incubator will not be used for the bacterial culture. 

[epcl_box type=”information”]Pro tips: The culture must be mixed gently twice a day by inverting the tube. [/epcl_box]

Pre-Harvesting: 

In the pre-harvesting stage, at the 70th hour of culture 100µL of 0.2% colcemid is added to the culture tube. 

Again incubate it for 2 hours more at the same condition. 

Harvesting: 

The culture tube is taken and centrifuged at 3500rpm for 8 to 10 minutes at room temperature. 

The supernatant is discarded and to the pellet, add 0.56% of KCl (called hypotonic solution, prewarmed at 37℃ ). 

The hypotonic solution is added up to the 8ml mark of the falcon tube., and mix it gently using the pasture pipette.  

Then incubate the tube in the prewarmed water bath at 37℃ for 15 to 17 minutes. 

Note: The hypotonic treatment is a very crucial step in the entire process, set your own time of incubation at which you will observe the good separation of chromosomes.  

The hypo-treatment step varies from lab to lab. 

Immediately after the hypo-treatment, add 2ml of chilled fixative (3 part methanol: 1 part glacial acetic acid). 

Mix it with the same pasture pipette until brown homogenizes mixture appears. 

The tubes are centrifuged at 2500rpm to 3500rpm for 8 to 10 minutes. 

Discard the supernatant and to the pellet add 8ml of chilled fixative again. 

Mix it properly and centrifuge at the same rpm and the same time as the last step. 

Repeat the fixative-centrifugation step until clear-white pellet appears. 

Once clear pellet appears, dissolve the pellet in the 2ml fixative. Now store the tube at 4℃ until the next step. 

Slide preparation and Giemsa staining:

Take a Greece free chilled slide and drop down 2 to 3 drops of the culture solution from ~2 feet hight. 

Chilled slide!!

Using a prechilled slide fix the chromosome faster. 

Heat fix the slide by heating it gently several times and stain it with Giemsa stain for 5 to 10 minutes.

Again!!

Heating will fix the chromosome faster, however, care must be taken while heating the slide, the chromosome components must not be burned. 

After staining, wash the slide with the running tap water and dry it. Observe the slide first in the 10X and then in the 100X under the microscope. 

Target 10 good metaphases from each slide for interpreting results. 

Read our amazing article on DNA: DNA story: The structure and function of DNA

Giemsa banding: 

• Three or four drops of the cell suspension dropped from the height of 2 feet.
• Age slide for 3 hours at 80℃.
• Stain slide in 2% Giemsa solution for 5 minutes.
• Deep the slide in the trypsin solution for 10 to 15 seconds and immediately wash it with the PBS.
• Again stain the slide in the Giemsa solution for some time (continuous stirred) and wash it with D/W.
• Air-dry the slide, the slide is ready for microscopic examination.

Conclusion: 

After reading this protocol, it seems like that the peripheral blood lymphocyte culture method for karyotyping is difficult, right!

But it is not. Even it is easier than the PCR. You just need to take care a bit more because you are dealing with cell culture. 

The only secret to success in karyotyping is to “maintain strict aseptic conditions.” 

 All the reagents are now commercially available in ready to use-form so don’t worry about it.