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397128, Life Technologies, Grand Island, NY, USA), 1.1 g of sodium bicarbonate, 5 ml of L-glutamine 200 mM 100X, 5 ml of antibiotic-antimycotic penicillin-streptomycin, and 50 ml of fetal bovine serum (JM Biosciences, San Diego, CA, USA).
Cells in diluent were added to 1 ml of PKH67 green fluorescent dye (2 µM) and incubated 5 min followed by a 1 min incubation with 2 ml of fetal bovine sera (FBS, National Veterinary Services Laboratory, Ames, Iowa) to adsorb the excess dye and stop further dye uptake by cells.
Germ tube test was performed by inoculating 0.5 mL of fetal calf serum with a loopful of yeast.
Administration of 100 IU (20 μg) Rh D immunoglobulin has been demonstrated to protect against 1 ml of fetal red cells, 500 IU (100 μg) should protect against FMH of up to 5 ml of fetal red cells and 1,500 IU (300 μg) Rh D immunoglobulin against FMH of approximately 15 ml of fetal red cells [ 20].
In addition, a maternal sample should be obtained approximately 1 hour after delivery and tested for evidence of a FMH in excess of 30 mL of fetal blood.
Staining was terminated by the addition of 4 ml of fetal bovine serum (FBS), incubation at 37°C for 30 min and washing three times in HBSS.
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The suspension was combined with 10 ml of 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle's Medium and cells were collected by centrifugation and resuspended in 10 ml of PBS.
Following centrifugation, the cells were resuspended in 0·5 ml of 2% fetal calf serum in HBSS with Ca/Mg and 10 mM HEPES, followed by incubation with normal mouse serum and fluorophore-conjugated monoclonal antibodies (above) for 1 h at 4°C.
They were washed twice by DMEM (containing 1500 mg/L glucose), then 2 ml DMEM free of fetal bovine serum and antibiotics was added into each well.
The resulting pellets were resuspended in 2 mL of PF10 (10% fetal bovine serum in phosphate-buffered saline), which typically yields 1 2 million total cells per mouse.
After 2 hours at 37°C, 5% CO2, 1 ml of DMEM, 2% fetal bovine serym (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin was added per well and incubated for 5 days.
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