Biomedical Engineering Reference
In-Depth Information
3.3. Isolation of CD71-Positive Umbilical Cord Blood Cells
The first steps in the actual in-cell PCR linker method described under Sub-
heading 3.3.1. outline the procedure for specific isolation of a subgroup of
cells in suspension.
3.3.1. Isolation of Specific Cell Populations With the Use
of Immunomagnetic Beads
1.
Process 1.0 or 0.5 mL of umbilical cord blood separately. Perform CD71 + cell
isolation from male or female samples alone or from mixtures (e.g., 1:1 or 1:9) of
male and female samples made up to a volume of 1.0 or 5.0 mL. Perform the
isolation of CD71 + cells as described by the manufacture of the immunomagnetic
beads.
2.
Briefly, add 1.2
10 7 anti-CD71 Dynabeads (Dynal A/S, Norway) to 1 mL cord
blood and incubate at 4°C with rotation for 20 min.
×
3.
Add washing buffer (1X PBS/2% FCS) in a volume of 2-3 mL and place the tube
in a magnetic particle concentrator (MPC) for 2-3 min.
4.
Discard the supernatant and wash the captured cells three times in washing buffer
and finally resuspend in 200
µ
L 1X PBS.
3.4. Fixation and Permeabilization of Cells in Suspension
Described as follows are the steps that can be used for fixation and perme-
abilization of the cells in suspension so the PCR reagents can enter the cells
( see Note 3 ).
1.
Place the CD71 + cells bound to Dynabeads in 200
µ
L 1X PBS in the magnet and
discard the supernatant.
2.
Add 100
L IntraStain Reagent A for fixation and resuspend the cells. Incubate
for 15 min at room temperature.
µ
3.
Add to the cell suspension 1.4 mL 1X PBS and mix carefully. Discard the super-
natant with the use of the magnet, and add 100
µ
L IntraStain Reagent B for per-
meabilization.
4.
After 15 min of incubation at room temperature, add 1.4 mL 1X PBS and place
the tube in the magnet and remove the supernatant.
5.
Wash the fixed and permeabilized cells three times in 100
µ
L 1X PCR-buffer.
3.5. In-Cell Linker PCR of a Y-Chromosome-Specific DNA Sequence
and an HLA-DPB1 DNA Sequence
The PCR steps in the in-cell linker PCR method are described under Sub-
headings 3.5.1.-3.5.3. First, the two PCR products of the Y-chromosome-
specific DNA sequence and the polymorphic HLA-DPB1 sequence are
co-amplified and linked together in a subsequent PCR process. Thereafter, the
two linked sequences are amplified further with the use of nested PCR.
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