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. 1.
Experimental Principles Mice bone marrow cells have a high degree of division activity and are in large numbers, so there is no need for in-body
culture
to directly observe the division phase of cells. Cells in the division phase are treated with akiylasin, which blocks the formation of spindle wire and stops cell division in the medium term, when chromosomes reach maximum contraction and have a typical form. Low seepage processing breaks cells and allows chromosomes to disperse. This method is used in the clinical study of leukemia, and can also be used to observe the toxic substances on the body's genetic material - chromosomal damage.
mouse chromosomes
2n-40,
were all
,
males with silk chromosomes
40
,
XY
, females
40
,
XX
.
2.
. Experiments
reagents
and equipment
microscope
,
ringer
water bath pot, low speed
centrifuge
, electronic
balance
, anatomical instruments (enamel anatomical disc, scissors, tweezers), syringes, needles,
test tubes
, test tube racks, droppers, load glass.
0.85
% physiological saline, fixed liquid
(
methanol: ice acetic acid is
3
:
1)
,
PBS (pH6.8)
,
Giemsa
Liquid, akilets
(
dissolved in
0.85
% physiological saline
) at a concentration of
100
μ
g/ml
)
,
0.075mol/L KCl
.
3.
. Experimental methods and steps
(
1
) autumn daffodil treatment:
3h
before taking bone marrow, the mouse abdominal cavity was injected with autumn daffodils at a dose of
100
μ
g
/
kg
animal weight.
(
2
) Take bone marrow: Kill the mice with spinal cord injury, then use scissors to cut open the skin and muscles on the thighs, remove the thigh bone, and clean the muscle residue attached to the bone with a small piece of gauze. Cut off the puffed joint heads at both ends of the collarbone, then use a syringe to absorb
5 ml 0.85
% physiological saline, insert it into one end of the collarbone, and rinse the bone marrow cells into the
centrifuge tube
of
10 ml
. Rinse repeatedly until the bone marrow cavity is white.
(
3
) low seepage treatment: the resulting cell suspension at
1000rpm
centrifugation
10min
, absorb the liquid, leave
0.2ml
sediment, plus
0.075mol/L KCL
solution (
37
degrees C) to
8 ml
scale, the cell sediment blown apart well, in
37
degrees C water bath low seepage
25min
.
(
4
) fixed: after low seepage treatment, immediately add
1 ml
newly provisioned and pre-cooled fixation liquid, pump evenly, and then
1000 rpm
centrifugation
10min
, discarded. Add the fixation fluid along the pipe wall to
6 ml
, and stationary fixation
10min
after blowing away the cells, i.e. for the first time. According to the above conditions centrifugation, go to the liquid, add the fixing liquid again to
6 ml
, for a second fixation.
10min
centrifuged to suck up the liquid, leave
0.2ml
sediment, and then add
4
drops of fixation to the sediment, flushed to make cell suspension.
(
5
) drops: take the pre-cooled slides in the refrigerator, from about
15cm
high to each slide drops
1
to
2
drops of cell suspension on the adhesive ice water slide, drying.
(
6
) staining: take the film flat on the glass plate, with
1
:
9 Giemsa
dye dye
20min
, water rinsed and dried.(
7
) observed the morphological characteristics of the chromosomes of mice and calculated the number of chromosomes
2n
of chromosomes.
.