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Primary Cell Culture
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For Chick Embryo


Materials required

 

  • Egg & Egg incubator
  • DBSS (Dissection Balanced Salt Solution)
  • BSS (Balanced Salt Solution)
  • DMEM (Dulbeco’s Modified Eagle Medium)
  • Forceps (Curved and Normal)
  • Petri plates (100 mm & 60 mm)
  • Test tubes and test tube rack
  • Pipettes & Micropipettes
  • Refrigerators
  • 370C Incubator

 

Procedure

 

  1. Take out a ten day incubated egg from 38.50 C egg incubator and swab it with 70% ethanol.
  2.  Crack the top with the back side of a sterile forceps and peel off the shell till the edge of the air sac.
  3.  Resterilize the forceps (i.e., dip them in alcohol, burn off the alcohol, and cool the forceps in sterile BSS), and then take the resterilized forceps and peel of the white shell membrane. Then we can see the chorioallantoic membrane and vasculature. Then remove the chorioallantoic membrane using the forceps.
  4.  Pierce the CAM with sterile curved forceps, and lift out the embryo by grasping it gently under the head. Do not close the forceps completely, or else the neck will sever; place the middle digit under the forceps and use the finger pad to restrict the pressure of the forefinger.
  5. Transfer the embryo to a 100 mm Petri dish and add 20 ml DBSS into it.
  6. Take two scalpels and dissect the embryo. Arrange the organ rudiments around the periphery of the dish.
  7. Take a test tube, open it and add 1 ml of 0.25% Trypsin into it. Transfer any of these tissues into the test tube containing trypsin using a forceps and close the tube.
  8. Incubate the tissue at 40C for 6- 18 hours in a refrigerator.
  9. After incubation, carefully remove the trypsin from the test tubes without disturbing the tissue.
  10. Again incubate the tissue in the residual trypsin for 15–20 min at 370 C.
  11. After incubation add 2ml of media to two 60mm Petri plate and to the test tube containing tissue and the residual trypsin. Then pipette up and down the media in the test tube to disperse the tissues.
  12. After dispersing the tissues using pipettes and transfer the tissue into Petri plates equally. Then mix the media in the Petri plate using a micro pipette.
  13. Incubating the cells in the CO2 incubator for 12 hours.
  14. After overnight incubation view the cells through the inverted microscope.
  15. Then check the confluency of plate and again incubate the cells in the CO2 incubator for 5-7 days to get a confluent culture.
  16. Again view the cells through the inverted microscope and maintain the primary cell culture.


For Zebra Fish Embryo


Materials required

 

  • Zebra fish embryo with chorion
  • Calcium free Reinger’s solution
  • Bleach Solution
  • Hank’s Saline solution
  • L- 15 growth media
  • Watchmaker’s
  • Forceps
  • Centrifuge tubes (15ml & 1.5 ml)
  • Cover glass
  • Micro pipettes & Pasture’s pipette
  • Petri plates ( 35 mm & 60 mm)
  • 28.50C incubator


Reagent preparation


1.    L- 15 growth media composition

 

 

Procedure

 

  1. Treat the zebra fish embryos with 0.5% bleach solution for 2 minutes before removing the chorions.
  2. Decant the bleach solution and rinse with 1000µl 10% sterile Hank's saline.
  3. Place the embryo in the 60mm tissue culture plate. Place it under a simple binocular microscope. By using watchmaker’s forceps make a tear in the chorion then make it upside down so that the embryo fall down from the chorion.
  4. Rinse embryos in sterile, calcium-free Ringer's solution.
  5. Transfer about 10-100 embryos in a single drop of calcium-free Ringer's solution to the center of a tissue culture plate and then drop a sterile cover slip onto the embryos and smash them.
  6. Transfer the suspension to a sterile centrifuge tube and spin at 300 x g for 7 min.
  7. Remove most of the supernatant, triturate the pellet through a sterile narrow-bore Pasteur pipette to resuspend the cells and then add several ml of growth medium.
  8. Centrifuge, 300 x g for 7 minute.
  9. Remove supernatant and resuspend in enough growth medium to make a final concentration of 15 fish per ml.
  10. Plate the cells and incubate overnight at 28.5°C.
  11. After the incubation view the cells under the inverted microscope.
  12. Then check the confluency of plate and again incubate the cells in the CO2 incubator for 5-7 days to get a confluent culture.
  13. Again view the cells through the inverted microscope and maintain the primary cell culture.

 

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