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  Section: Biotechnology Methods » Molecular Biology
 
 
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Culturing Peripheral Blood Lymphocytes

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Principle
Peripheral blood lymphocytes are incubated in a defined culture medium supplemented with serum, phytohemaglutinin, and other additives for the purpose of preparing metaphase chromosomes.


Time Required

72 to 96 hours.


Special Reagents
  1. McCoy 5A (Tissue support center)
  2. Fetal Bovine Serum (Tissue support center)
  3. L-Glutamine, 200 mM, 100X, (Tissue support center)
  4. Phytohemaglutinin
  5. Gentamicin Reagent Solution (50 mg/mL), liquid
  6. Vacutainer, green top, sodium heparin
  7. Heparin, sodium salt 300 USP

Safety Considerations

Because we do not test the cell lines or blood samples, always work under the assumption that the cells carry infectious agents, e.g., HIV virus, hepatitis B, etc. Keep the samples isolated, work only in the biological safety hoods, and always wear gloves. Autoclave all waste materials.


Procedure
Day 1
  1. Collect blood by venipuncture in 1 or 2 7-mL sterile venipuncture tubes coated with sodium heparin. Rotate the tubes to prevent clotting. Label tubes with individual’s name, date, and time of drawing the specimen.
    Store at room temperature (never place in refrigerator or on ice).
  2. Label 8–10 15-mL centrifuge tubes, then pipette 10 mL McCoy 5A growth medium to each labeled tube. Add 9–12 drops of whole blood to each, using a sterile 5 ¾" Pasteur pipette. Cap the centrifuge tubes tightly and mix gently by inverting tubes. Loosen the caps on the 15-mL tubes and incubate at 37°C in a centrifuge rack positioned at a 45° angle for approximately 72 hours.
Days 2-3
  1. Mix the blood by inverting the tubes twice daily during the 3-day incubation period.

    If clumps form at the bottom of a culture tube, 5–10 drops of heparin (300 USP) can be added with a 1-cc syringe.

Solution
  • McCoy 5A growth medium:

    McCoy 5A
    Phytohemaglutinin
    L-Glutamine
    Fetal bovine serum
    Gentamicin
    1000.0 mL
    10.0 mL
    10.0 mL
    100.0 mL
    1.2 mL

  • Filter sterilize with 0.22 mm cellulose acetate membrane; store medium at 4°C for up to 2 weeks, or freeze medium in 50-mL centrifuge tubes at –80°C for up to 1 year.
  • Phytohemaglutinin, M form, PHA, lyophilized
  • Rehydrate with 10 mL sterile double distilled water (ddH2O). Store at
  • 2°C–8°C. After reconstitution, the phytohemaglutinin solution is good for 14 days.
  • Heparin, sodium salt 300 USP: Rehydrate with 5 mL of McCoy growth medium. Store at 4°C for up to 2 weeks.
 
     
 
 
     




     
 
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