Lymphocyte Preparation: Preparing the Culture

Lymphocyte Preparation: Preparing the Culture
  1. Draw 8-10 mL peripheral blood into a 10 mL green-top vacutainer tube containing sodium heparin preservative. Mix well.
  2. Aseptically transfer the contents of the vacutainer tube into a 15 mL centrifuge tube.
  3. Centrifuge the sample at 2000 rpm for 10 minutes in a table-top centrifuge.
  4. Carefully remove the plasma layer using a Pasteur pipet. Discard the plasma layer following your laboratory procedure for disposal of biological material.
  5. Remove the buffy-coat by gently circling the layer while drawing the WBCs into a Pasteur pipet. Some RBCs and plasma will be drawn up also;minimize the number of RBCs included.
  6. Expel the buffy-coat into a 15 mL centrifuge tube that contains 1.5 mL of cRPMI.
  7. Aliquot appropriate volume of WBC suspension to 25 cm2 flasks containing 10 mL cRPMI. 
  8. Add 0.2 mL of PHA to each flask; tighten cover and then loosen slightly to allow CO2 to enter flask.
  9. Incubate the culture:

for use in FISH slide preparation ...

This method produces chromosomes with a band resolution of 400-450 and is sufficient for most FISH applications. 

    • Incubate culture in 37 ±1 °C, 5% CO2 environment for 72-96 hours.

for use in CGH slide preparation ...

These additional steps increase chromosome elongation and mitotic index. For CGH, the chromosomes should have a band resolution of 400-550.

    • Incubate culture in 37 ±1 °C, 5% CO2 environment for 48-72 hours.
    • Add 0.2 mL of thymidine solution to each flask and gently mix.
    • Incubate culture for 14-18 hours.
    • Transfer the culture to a 15 mL centrifuge tube.
    • Centrifuge at 1000 rpm for 10 minutes.
    • Decant supernatant and add 10 mL of PBS.
    • Centrifuge at 1000 rpm for 10 minutes.
    • Decant supernatant and add 10 mL of cRPMI to each tube.
    • Incubate culture for 4-5 hours.
 
 
 
 
Harvesting the culture
  1. Draw 8-10 mL peripheral blood into a 10 mL green-top vacutainer tube containing sodium heparin preservative. Mix well.
  2. Aseptically transfer the contents of the vacutainer tube into a 15 mL centrifuge tube.
  3. Centrifuge the sample at 2000 rpm for 10 minutes in a table-top centrifuge.
  4. Carefully remove the plasma layer using a Pasteur pipet. Discard the plasma layer following your laboratory procedure for disposal of biological material.
  5. Remove the buffy-coat by gently circling the layer while drawing the WBCs into a Pasteur pipet. Some RBCs and plasma will be drawn up also;minimize the number of RBCs included.
  6. Expel the buffy-coat into a 15 mL centrifuge tube that contains 1.5 mL of cRPMI.
  7. Aliquot appropriate volume of WBC suspension to 25 cm2 flasks containing 10 mL cRPMI.
  8. Add 0.2 mL of PHA to each flask; tighten cover and then loosen slightly to allow CO2 to enter flask.
  9. Incubate the culture:

for use in FISH slide preparation ...

This method produces chromosomes with a band resolution of 400-450 and is sufficient for most FISH applications. 

    • Incubate culture in 37 ±1 °C, 5% CO2 environment for 72-96 hours.

for use in CGH slide preparation ... 

These additional steps increase chromosome elongation and mitotic index. For CGH, the chromosomes should have a band resolution of 400-550.

    • Incubate culture in 37 ±1 °C, 5% CO2 environment for 48-72 hours.
    • Add 0.2 mL of thymidine solution to each flask and gently mix.
    • Incubate culture for 14-18 hours.
    • Transfer the culture to a 15 mL centrifuge tube.
    • Centrifuge at 1000 rpm for 10 minutes.
    • Decant supernatant and add 10 mL of PBS.
    • Centrifuge at 1000 rpm for 10 minutes.
    • Decant supernatant and add 10 mL of cRPMI to each tube.
    • Incubate culture for 4-5 hours. 
 
 
 
 
Fixing the Sample
  1. Slowly add 2 mL of Carnoy's fixative down the inside of each centrifuge tube.
  2. Gently tap the centrifuge tube to mix the cell pellet suspension with the fixative.
  3. Slowly add another 6 mL of Carnoy's fixative to each tube.
  4. Tightly cap each tube and invert several times to mix. 
  5. Let the suspension stand 5 minutes at ambient temperature, then centrifuge at 1000 rpm for 10 minutes.
  6. Aspirate the supernatant to the 1.5 mL mark on the centrifuge tube, taking care to avoid the cell pellet
  7. Gently tap the centrifuge tube to dissociate the pellet.
  8. Perform the following steps three times:
    • Slowly add 10 mL of Carnoy's fixative to each centrifuge tube.
    • Tightly cap each tube and invert to mix. 
    • Centrifuge the suspension at 1000 rpm for 10 minutes.
    • Aspirate the supernatant to the 1.5 mL mark on the centrifuge tube, taking care to avoid the cell pellet.
    • Gently tap the centrifuge tube to dissociate the pellet.
  9. Slowly add Carnoy's fixative to each suspension to produce a slightly cloudy suspension and gently mix.

 

Note: You add fixative to the cell pellet until the suspension is slightly cloudy; the amount of fixative needed will depend on the size of the cell pellet. 

 

Cells may be stored at 4 °C in tightly-capped centrifuge tube until ready to use. If slides will not be prepared within 7 DAYS, bring the total volume to 10 mL and store at -20 °C. Repeat the wash described in steps 8a-8e two times prior to preparing the slides.

 
 
 
 

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