I. Purpose:
Amniotic fluid may be used for prenatal diagnosis of aneuploidy or other structural abnormalities.
II. Culture Procedure:
A. Aseptic technique must be used when setting up the cultures under the laminar flow hood.
B. Cultures are grown in a mixture of CHANG and F-10. The media must be fresh (less than 4 days old), prewarmed, and at pH 7.0-7.5. All cultures are incubated in a wet, 5% CO2 incubator at 37 C.
C. Specimens are usually received in two 15 ml conical centrifuge tubes. If the sample is received differently, transfer to two tubes. Centrifuge the tubes for 6-8 minutes at 150 x g (900 rpm). Leaving 0.5 ml, transfer supernatant to a 15 ml centrifuge tube (this will be frozen for back-up AFP studies). If specimen is needed for prenatal tests, save 2ml of supernatant in a vial (like the ones used for tissue transport). Parafilm the vial, place in specimen bag and put in fridge. Please notify Kate,Sue, or Dr. Sekhon of sample to go to SLH.
D. Label 30mm petri dishes w/coverslips with lab#, patient name, AF, date set, and culture (A or B). Routinely, set 4 coverslips per tube. For small pellets, set 3 coverslips per tube. If only one tube was sent, set 6 coverslips from that one tube.
E. Resuspend pellet in media (4 coverslips, add 1.5 ml ; 3 coverslips, add 1.0 ml of media). Add 0.5 ml to each coverslip, being careful to keep it on the coverslip. Do the same for each tube of amniotic fluid. Set-up day is day 0. Place "A" coverslips into incubator #3, and "B" coverslips into incubator #2.
F. On day 2 flood the coverslips with 1.5 ml of media. A and B cultures should be fed with appropriate bottles of media, each labelled A or B. On day 4 feed with 0.5 ml of media.
G. Starting on day 4, check for growth. Ideally, this will be as individual colonies dispersed over the coverslips. On day 5 or 6, depending on the number and size of colonies, media should be changed and reset. Media should be changed when coverslips can be harvested the following day. One coverslip should be reserved from harvest for possible subculturing. (If, on day 6, coverslips are not ready for a full media change, carefully aspirate off 1 ml of media per coverslip, discard, and refeed with 1 ml fresh media using the appropriate A or B media bottle. Follow these coverslips until ready for a full media change and reset.)
H. Change the media by using a sterile pipette to transfer the suspension into a sterile 15 ml conical tube. (All 'A'coverslips in one tube, and all 'B' coverslips into a second tube.) Rinse the coverslips using 2 ml fresh media (using the appropriate A or B media). The rinse media should also be saved in the 15 ml tube. Rinse twice if necessary.
Add 2 ml media to each dish and place in the incubator.
RESET PROTOCOL
1. Centrifuge for 6 - 8 minutes at 150 x g (900rpm). Discard the supernatant. For each tube, resuspend the pellet using1.0 ml of fresh media and plate onto 2 labeled coverslips. (if the pellet is large, resuspend in 1.5 ml of media and plate onto 3 coverslips). Coverslips should be labeled with lab number, AF, culture A or B, date and 'reset'.
2. On day 2, flood the coverslips with 1.5 ml media again using the appropriate A or B media.
3. On day 3, feed the coverslips with 0.5 ml media. Also check them for possible media change. (Resets grow faster than primaries.)
4. On day 4, check for a media change and harvest.. Discard this media. No reset is required.
I. Coverslip Subcultures - if necessary, coverslip cultures may be split (subcultured) onto more coverslips to improve growth. This is done by the following procedure:
- Remove and discard media from coverslips
- Add 0.2 ml trypsin, let sit 20 sec (rocking gently)
- Remove and discard trypsin
- Add 0.2 ml trypsin, incubate 5 min
- Gently knock off cells
-Using 2 ml media (Chang/F-10), wash all dishes and transfer to a sterile 15 ml conical centrifuge tube.
-repeat with an additional 2 ml media
-Centrifuge tube
-Resuspend in 0.5 ml media per coverslip to be set.
-Flood coverslips at end of day with 1.5 ml of media and harvest one to two days following setup
III. Harvest Procedure:
A. Starting on day 5 check the coverslips to see if they are ready for harvest. There should be multiple colonies with rounded, dividing cells on each coverslip. If the colonies are allowed to grow too large they may grow too dense or into one another and there will be too much cytoplasm for good spreading and banding of the chromosomes. If the colonies are too small, insufficient metaphases may be found.
B. Add 20 µl Ethidium Bromide working solution to each dish, incubate for 40 minutes. Add 40 µl of colcemid working solution, incubate for another 20 minutes.
C. Ten minutes before end of incubation, start up TECAN harvester so that it will be ready.
D. Harvest using TECAN.
E. Drying conditions are very important for the proper spreading of metaphases. Using the TECAN dry program, remove fixative from the dish. Using the aspirator, remove almost all the fix, going around the edge of the coverslip. Allow to dry in the Percival Scientific drying chamber set at 35.0% relative humidity, 28.0°C, fan speed 72 - 75%. If the coverslip dries too rapidly, all the cells will be trapped in the membranes. If it dries too slowly, the chromosomes will float away from the metaphase.
F. Remove the coverslip from the petri dish, keeping it right-side up. It helps to hold the dish with your thumb and middle finger and bend up the bottom of the dish with your index finger so that the coverslip is lifted up. Mount the coverslip on a labeled microscope slide using a drop of mounting media. Place only one coverslip per slide, properly labeled with patient number, A or B culture, AF, and 'reset' if necessary.
G. Allow the slides to dry for at least 30 minutes at room temperature. Bake at 90°C for 30 - 60 minutes. Slides are now ready for G, Q, or C banding. Metaphases are resistant to trypsinization and also tend to require more stain than other types of specimens.
V. Solutions:
Colcemid working solution: 10 mcg/ml Colcemid in Hank's Balanced Salt Solution, store at 4 C.
Complete CHANG/F10 media: 45 ml Chang basal media B, 42 ml Nutrient Mix F-10, 5 ml Chang supplement A, 8 ml Fetal calf serum, 1 ml Penicillin/Streptomycin, solution, 1.1 ml L-Glutamine solution, store at 4 C.
Ethidium Bromide Working Solution: 2 mg/ml Ethidium Bromide in RPMI-1640, stored in dark.
Fixative: 30 ml Methanol and 10 ml Glacial Acetic Acid, prepared fresh.
Hypotonic solution: 0.8%NaCitrate/0.075 M KCl: 1.6g NaCitrate, 1.68g Potassium Chloride dissolved in 500ml dH2O.
1X Trypsin EDTA solution: 10ml stock trypsin solution (10X trypsin in saline), 0.1g EDTA (disodium salt), dissolved in 490ml Hank's Balanced Salt Solution. Sterile filtered, 50ml aliquots. Store frozen.
VI. Reagents::
Chang media: IRVINE cat# T100-018, liquid and frozen supplement, 100 ml bottle, store supplement frozen, basal media at 4 C.
Colcemid: GIBCO cat#15210-040. 10 mcg/ml in Hank's Balanced Salt Solution, 10 ml bottle, store at 4 C.
Ethidium Bromide: SIGMA cat# E-8751. 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide, 250 mg bottle.
Fetal calf serum: HYCLONE/STERILE SYSTEMS INC. cat# A-1111-D. defined fetal bovine serum, 100 ml bottle, store frozen.
Glacial Acetic Acid: FISHER cat # A38-500. Acetic acid, Glacial ACS (Aldehyde free), 500 ml bottle.
L-Glutamine: GIBCO cat# 320-5030. L-Glutamine solution 100X (200 mM), 20 ml bottle, store frozen.
Methanol: FISHER cat #A412-500. Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free, 500 ml bottle.
Nutrient Mix F-10: GIBCO cat# 81200-040. Nutrient Mixture F-10 (HAM) powdered form, prepared to instructions, 10 x 1 liter package.
Penicillin/Streptomycin: GIBCO cat# 600-5140. Penicillin/Streptomycin solution, 10,000 units/ml / 10,000 mcg/ml, 20 ml bottle, store frozen.
Potassium Chloride: COLUMBUS CHEMICAL INDUSTRIES, INC. ACS granular, 500 g lots.
Sodium Citrate: UW-Stores, cat# 2588. Crystal, ACS, Na3C6H507.2H20, 500g bottle
Trypsin Stock Solution(1X): GIBCO cat# 25300-013. Trypsin 2.5% (10X) in saline. Store frozen
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