CN104178451B - From milk, separate and cultivate method and the special culture media of cow mammary gland epithelial cells - Google Patents
From milk, separate and cultivate method and the special culture media of cow mammary gland epithelial cells Download PDFInfo
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Abstract
The invention discloses a kind of method and special culture media separating and cultivating cow mammary gland epithelial cells from milk.The present invention discloses a kind of method separating from cow's milk juice and cultivating galactophore epithelial cell, comprises the steps: to select somatic number in cow's milk juice to be less than 5 × 104Individual/ml and be in the ox of lactation middle and later periods, gathers cow's milk juice；Separate and cultivate the galactophore epithelial cell in cow's milk juice.Method disclosed by the invention by Cow product record limits suitable milk sampling ox condition only and determine the collection phase as the lactation middle and later periods, by hand milking's method, easy, quick, economic acquisition milk sample, and separate, cultivate galactophore epithelial cell, reliable results, stable, simple to operate, being suitable for Routine Test Lab experiment needs, low cost, cell state are good, simultaneously compared with tissue mass cell culture, only need to obtain milk sample, only not damaging ox, simple to operate, the scope of application is wider.
Description
Technical field
The present invention relates to a kind of method and special culture media separating and cultivating cow mammary gland epithelial cells from milk, belong to
In biological technical field.
Background technology
Breast is dam distinctive nurture organ, be full of in lactation period mammary gland abundant acinus and conduit (Cui Lili,
2006).Mammary gland is the spherical structure surrounded by outer layer myoepithelium and internal layer secretory epithelial cells, is connected with conduit, its
Middle secretory epithelium is the elementary cell of acinus interior synthesis milk composition, is the basis of its enforcement lactation function.Therefore, it is
Research milk composition synthesis and changing mechanisms, mammogenesis and change and tumor of breast are formed, the correlation factor of canceration and
The indispensable experiment material (cuckoo etc., 2007) of gene.
At present, existing more than 100 year of the cultivation history of galactophore epithelial cell, as a kind of well differentiated adult cell,
It often occurs the dedifferentiation of form and characteristic after cultured in vitro a period of time, tends to become fibroblast-like state
(Ebner etc., 1961).Limited diploid cell, owing to departing from normal vivo environment, gradually takes off after original cuiture
The phenomenon of differentiation is the most common.In the face of this problem, researcher with gene transfer technique by SV40, proto-oncogene
With in the exogenous origin gene integrator such as Human telomerase reverse transcriptase gene to limited cellular, the conversion cell (pottery of being immortalized property
Modest etc., 2001).Immortality cell while remain the part biological nature the most similar to cells of origin and phenotype,
Also show some convert and purify the new feature after cultivating.Foreign gene random integration is to the position of host cell gene group
Putting uncertain, its insertion point may have influence on the function of the original gene of cell, and clone cell is owing to lacking cell interaction
Impact also show the feature made new advances.Reaction that the factor is stimulated by existing immortalization galactophore epithelial cell system and milk composition
Expression there are differences, these uncertainties make it apply to be restricted.
Due to many uncertainties of immortalized cells, primary cell line more can represent internal organ specificity function and
Signal transduction pathway.The galactophore epithelial cell of general in vitro culture can stably cultivate some months, can meet cell experiment
The needs in cycle.Therefore, most biological experiments or the galactophore epithelial cell (Pantsckenko of first-selected original cuiture
Deng, 2000).Primary cell loses the restricted characteristic that some are original after repeatedly passing on, at growth rate, breast
There are differences with internal cell in juice synthesis, the reaction that the factor is stimulated and sensitiveness.But, this problem can be led to
The cell crossing low tempertaure storage fresh separated solves, to cultivate cell use freeze-thaw technology (Bramley etc.,
1982), to keep its original cell characteristics, allow and meet the primary cell of Research Requirements and passage cell can be grown
Kubo is deposited, and makes the feature analyzing same primary characteristic cell under different experimental conditions be possibly realized.Meanwhile, can pass through
Add some bioactie agents (insulin, hydrocortisone, EGF, transferrins etc.) and promote thin
Born of the same parents' propagation and maintenance cell characteristics, delay cell dedifferentiation.
The method that current domestic cultivation primary mammary epithelial cells generally uses is tissue mass cell culture, and this method is passed through
Living body puncture or butcher acquisition biological tissue, on the one hand the infringement to ox body is relatively big, the cost of buying healthy adult ox is high；
On the other hand, many acquisitions is the biological tissue eliminating ox, it is difficult to obtain the tissue sample of high yield ox.
Summary of the invention
It is an object of the invention to provide a kind of method separating and cultivating cow mammary gland epithelial cells from milk and special training
Support base.
The present invention provides a kind of method separating from cow's milk juice and cultivating galactophore epithelial cell, comprises the steps: choosing
Select somatic number in cow's milk juice and be less than 5 × 104Individual/ml and be in the ox of lactation middle and later periods, gathers cow's milk juice；Separate
And cultivate the galactophore epithelial cell in cow's milk juice；
The described lactation middle and later periods is 150-250d after calving；
Before during described collection cow's milk juice, 50-100ml cow's milk juice is not collected.
In said method, described separation and cultivate the galactophore epithelial cell in cow's milk juice include separate and cultivate primary cow's milk
Glandular epithelium, more primary bovine mammary epithelial cell is carried out Secondary Culture, obtain the bovine mammary epithelial cell passed on.
In any of the above-described described method, described primary bovine mammary epithelial cell and the bovine mammary epithelial cell passed on are cultivated
Time the culture medium that uses be prepared as follows: be 9:1 by DMEM/DF12 culture medium and FBS according to volume ratio
Mixing, add final concentration of 1 × penicillin, final concentration of 1 × streptomysin, the pancreas islet of final concentration of 5ug/mL
Element, the transferrins of final concentration of 5ug/mL, the hydrocortisone of final concentration of 5ug/mL, final concentration of 10ng/mL
EGF, the progesterone of final concentration of 1ug/mL, the recombinant bovine galactagogin of final concentration of 10ng/mL；
Described DMEM/DF12 culture medium is purchased from Gibco, and catalog number is C11330500BT；
Described final concentration of 1 × penicillin and final concentration of 1 × the mother liquor of streptomysin be concentration be 100 × mould
The dual anti-solution of element/streptomysin；
Described concentration is 100 × the trade name penicillin/streptomycin of the dual anti-solution of penicillin/streptomycin dual anti-molten
Liquid (100 ×), purchased from Gibco, catalog number is 15140-122；
Described recombinant bovine galactagogin is purchased from Prospec, and catalog number is CYT-511.
In any of the above-described described method, described ox is milk cow.
In any of the above-described described method, described milk cow is Holstein cow.
A kind of culture medium falls within protection scope of the present invention, is prepared as follows: by DMEM/DF12 culture medium
Be 9:1 mixing with FBS according to volume ratio, add final concentration of 1 × penicillin, final concentration of 1 × streptomysin,
The insulin of final concentration of 5ug/mL, the transferrins of final concentration of 5ug/mL, the hydrogenation of final concentration of 5ug/mL can
Pine, the EGF of final concentration of 10ng/mL, the progesterone of final concentration of 1ug/mL, final concentration of 10ng/mL
Recombinant bovine galactagogin；
Described DMEM/DF12 culture medium is purchased from Gibco, and catalog number is C11330500BT；
Described final concentration of 1 × penicillin and final concentration of 1 × the mother liquor of streptomysin be concentration be 100 × mould
The dual anti-solution of element/streptomysin；
Described concentration is 100 × the trade name penicillin/streptomycin of the dual anti-solution of penicillin/streptomycin dual anti-molten
Liquid (100 ×), purchased from Gibco, catalog number is 15140-122；
Described recombinant bovine galactagogin is purchased from Prospec, and catalog number is CYT-511.
The method that the present invention provides limits the condition of suitable milk sampling ox by Cow product record and determines collection
Phase is the lactation middle and later periods, by the method for hand milking, and easy, quick, economic acquisition milk sample, and separate,
Cultivate galactophore epithelial cell, reliable results, stable, simple to operate, it is suitable for Routine Test Lab experiment needs, low cost,
The cell state obtained is good.The method of the present invention is compared with tissue mass cell culture, it is only necessary to obtain milk sample, to ox only
Not damage, simple to operate, the scope of application is wider.
Accompanying drawing explanation
Fig. 1 is the form of the primary mammary epithelial cells separated from milk.
Fig. 2 is bovine mammary epithelial cell growth curve.
Fig. 3 is the Keratin 18 coloration result of galactophore epithelial cell.
Fig. 4 is the RT-PCR testing result of milk composition related gene in galactophore epithelial cell.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
DMEM/DF12 culture medium is purchased from Gibco, and catalog number is C11330500BT.
Hyclone (FBS) is purchased from Gibco, and catalog number is 10099-141.
The dual anti-solution of penicillin/streptomycin (100 ×) is purchased from Gibco, and catalog number is 15140-122.
Insulin (ox, cell grade) is purchased from Sigma, and catalog number is I5500.
Transferrins is purchased from Sigma, and catalog number is G7250.
Hydrocortisone is purchased from Sigma, and catalog number is H0888-1G.
EGF is purchased from Gibco, and catalog number is PHG6045.
Progesterone is purchased from Sigma, and catalog number is P8783.
Recombinant bovine galactagogin is purchased from Prospec, and catalog number is CYT-511.
The preparation of the complete medium used in following embodiment: be 9 according to volume ratio by DMEM/DF12 and FBS:
1 mixing, add the dual anti-solution of penicillin/streptomycin to the final concentration of antibiotic be 1 ×, add final concentration following
Each factor: insulin (5ug/mL), transferrins (5ug/mL), hydrocortisone (5ug/mL), EGF
(10ng/mL), progesterone (1ug/mL), recombinant bovine galactagogin (10ng/mL), this culture medium is thin for breast epithelium
The cultivation of born of the same parents.
Nonimmune lowlenthal serum is purchased from Fuzhou Maixin biotechnology Development Co., Ltd.
Anti-cytokeratin 18 antibody is purchased from abcam, and catalog number is ab668.
Fluorescently-labeled two anti-(alexa488goat anti-mouse IgG (H+L)) purchase Proteintech
(PTG), catalog number is SA00006-1.
BSA antibody diluent is purchased from Beijing CoWin Bioscience Co., Ltd., and catalog number is CW2340.
Embodiment 1, the selection of ox of suitable sampling and the collection of milk sample and preservation
One, somatic number in cow's milk is selected to be less than 5 × 10 according to the record of production4Individual/ml's (avoiding recessive mastitis)
The Holstein cow being in lactation middle and later periods (after calving 150～250d) of udder health, adopts twice milk for one day, once early
On, in an afternoon, the most only receive a milk.Manual milking when adopt milk in the morning for the first time, typically collects 150mL
The milk of left and right to sterile centrifugation tube (before collection, with collected ox the nipple of bulk cotton ball soaked in alcohol wiping, first three
Milk about 50-100ml removes and does not receives, to reduce the pollution to milk at nipple of the outer derived bacterium)), only change ox simultaneously
When, change disposable glove, it is to avoid milk is formed exogenous pollution.
Two, after milk is collected, after the complete centrifugal mouth of pipe of cotton ball soaked in alcohol wiping and lid, after sealing with sealed membrane, carry out
Mark, is maintained at the temperature of milk between 4-37 DEG C, it is ensured that sample takes back laboratory at about 2h, in order to carry out
Subsequent treatment.
The separation of primary mammary epithelial cells and collection in embodiment 2, cow's milk
One, milk embodiment 1 obtained 1000r/min in normal temperature centrifuge is centrifuged 20min, centrifugal rear ox
Milk is divided into two-layer, and upper strata is the butterfat of thickness, and lower floor is muddy liquid.Now cell concentrates on bottom centrifuge tube,
Remove upper strata butterfat as far as possible, retain 10mL bottom troubled liquor, reduce bottom liquid as far as possible and rock.
Two, adding the aseptic PBS of equivalent in bottom troubled liquor, after mixing, 1000r/min is centrifuged 10min,
Remove supernatant liquid, continue to retain 10mL bottom liquid.Repeat this step 3～5 times, until bottom liquid clarification.
Three, the bottom liquid that step 2 obtains being proceeded to 15mL centrifuge tube, 15000r/min is centrifuged 10min, inhales
Abandon supernatant, retain 1mL supernatant.
Four, the upper cleer and peaceful centrifuge tube bottom precipitation retained with pipettor mixing, moves to T25 Tissue Culture Flask, has added
Full medium culture galactophore epithelial cell.
Five, the complete medium renewed every other day.Before changing liquid, after the old culture medium of sucking-off, blake bottle is tilted, at bottleneck
Place is slowly added to PBS, makes PBS slowly cross cell surface along inclined plane, then removes cleaning fluid PBS, repeat 1～2
This operation secondary, removes acellular components the most adherent in blake bottle, adds new complete medium and continues to cultivate.
Embodiment 3, the original cuiture of galactophore epithelial cell, pass on, frozen and recovery
One, the complete medium that cell embodiment 2 cultivated renewed every 3 days is once, until bigger cell gram
Grand formation.
The form of the primary mammary epithelial cells separated from milk is as shown in Figure 1.
In Fig. 1, A1 is to cultivate the form by the 5th day, after single primary mammary epithelial cells is adherent；A2 is that cultivation is arrived
5th day, form, after single primary mammary epithelial cells propagation, the clone (100 ×) being about 10 cells.B1 and B2
For cultivating by the 14th day, the big cell colony (100 ×) that monoclonal is formed.After C is for cultivating 20 days, part
Intracellular there is drop (100 ×).After D is for cultivating 20 days, the part cell occurred in cell colony surrounds
Cavity (100 ×).
The galactophore epithelial cell that the centrifugal primary mammary epithelial cells primary vaccination obtained is lived the most afterwards from cow's milk juice i.e. pastes
Wall, now cell culture medium left floating the impurity such as substantial amounts of albumen, lipid and dead cell, and cell culture medium is micro-
Microscopic observation suspended components is more, should change liquid the most afterwards in time, remove the most adherent composition, prevent impurity effect thin
Intracellular growth.The cell quantity obtained in milk is less, and the first day is typically difficult to find out attached cell, waits to cultivate 3～5d
After, unicellular adherent sprawl after, form is clear and starts propagation, it is possible to observe that individual cells or a few cell are constituted
Little clone's colony, cell becomes round pie, short fusiformis or polygonal (such as A1 and A2 in Fig. 1).Continue to cultivate,
Cell starts to breed in a large number, and cell colony increases (such as B1 and B2 in Fig. 1) and mutually collects.Subsequently, part is thin
Born of the same parents there will be drop-wise structure (as shown in C arrow in Fig. 1), intensive cell colony occurs the cavity that cell surrounds
(as shown in D arrow in Fig. 1).
The cell separated from milk is epithelial types, does not has fibroblastic pollution.
Two, after relatively maxicell Clone formation (about the 21d that primary vaccination is cultivated), galactophore epithelial cell can be carried out
Pass on, frozen and recovery experiment.
(1) passage, operates as follows: with 0.25% pancreatin/EDTA digestive juice vitellophag 3-5min at 37 DEG C
After, sample loading gun is drawn digestive juice and is repeatedly purged cell monolayer surface, collects digestive juice to containing equivalent complete medium
Centrifuge tube terminates digestion, repeats aforesaid operations 1 time, collect residue attached cell.By the cell of collection 1000
R/min is centrifuged 5min, retains precipitation, is inoculated in new culture dish, adds new complete medium, inoculation
Density can form 40% cell concentration collecting rate by 24h and calculate (cell density suitable when holding is passed on, cell density mistake
Time low, cell proliferation rate is the most slack-off), change liquid after incubated overnight, within the most every 3 days, change a subculture.
(2) cell cryopreservation, operates as follows: collect, with 0.25% pancreatin/EDTA digestive juice, the biography that step () obtains
The cell monolayer of the exponential phase in generation, centrifugation, add 1mL complete medium re-suspended cell, take the most resuspended
After liquid suitably dilutes, carry out blood count, with frozen protection liquid (DMEM/F12:FBS:DMSO volume ratio is 7:
2:1) diluting cells is dispensed in the cryopreservation tube of 2mL to the suitable order of magnitude, every 1mL, general 10cm2Plate
The cell of results can be managed with frozen 3-5.Freeze-stored cell employing gradient cooling method: 4 DEG C of 10min ,-20 DEG C of 30min,
-80 DEG C overnight, and cell proceeds to liquid nitrogen container Long-term Cryopreservation the most afterwards at ultra low temperature freezer.
(3) cell recovery: take out frozen passage cell from liquid nitrogen container, shakes in the water-bath of 37 DEG C, promotees
Making cell thaw rapidly, cell suspension aseptically proceeds to centrifuge tube, 1000r/min is centrifuged 5min, protects
Cell is stayed to precipitate, 1mL complete medium re-suspended cell, it is inoculated in T25 blake bottle, 37 DEG C are incubated, overnight
Change culture medium.Within the most every 3 days, change 1 new culture medium.
Embodiment 4, galactophore epithelial cell Property Identification
One, growth characteristics are identified
Cell growth curve is drawn: be inoculated in 24 well culture plates by the cell of Secondary Culture to the 5th generation, and every hole adds
5×104Individual cell.Arrange 14 groups altogether, often 3 repetitions of group, 1 group of cell of 1～14 day every day of digestion collection after inoculation,
3 porocytes often organized are mixed, adds 400 μ L PBS mixings after centrifugation, use blood cell counting plate weight
Counting 5-6 time again.Using incubation time as abscissa, the average of the every porocyte calculated with every day, as ordinate, is painted
Make the bovine mammary epithelial cell growth curve of 14 days, as shown in Figure 2.
Cell growth curve is usually used in the growing multiplication ability of measure of cell.The growth incubation period to be experienced of passage cell is (thin
Born of the same parents slowly breed), exponential phase of growth (cell fast breeding), plateau (cell quantity is constant) and decline phase are (thin
Born of the same parents are dead) 4 parts.
Fig. 2 shows, the bovine mammary epithelial cell of in vitro culture is in plateau in front 7d, cell poor growth,
To adapt to new culture environment, initially enter exponential phase of growth from the 8th, after 13d, enter the plateau of proliferation slowed down,
Result shows, the growth curve of galactophore epithelial cell is typical S type.
Two, immunofluorescence (IF) dyeing identification of cell Keratin 18
Keratin is the distinctive cytoskeletal protein of epithelial cell, by the table of the intracellular Keratin 18 of Immunofluorescence test
Reach, to judge the epithelium characteristic of cell.Concrete operations are as follows:
(1) being inoculated by suitable number by galactophore epithelial cell, inhale every other day and abandon nutrient solution, PBS uses body after flushing three times
The PBS solution of long-pending percentage composition 2%～4% formaldehyde fixes 15min, obtains the cell fixed；
(2) by the cells rinsed with PBS that fixes 3 times, each 5 minutes, nonimmune lowlenthal serum is dripped (interior
It is 0.3%Triton X-100 containing volumn concentration), cover cell liquid level 2～3mm, room temperature places 60min,
Get rid of surplus liquid；
(3) anti-Anti-cytokeratin 18 antibody of dropping CK18 is (with BSA antibody diluent according to volume
Dilute than 1:500) 50 μ L, 4 DEG C are overnight.
(4) after overnight, wash 3 times with PBS, each 5min, drip fluorescently-labeled two anti-alexa488
Goat anti-mouse IgG (H+L) (dilutes according to volume ratio 1:200 with BSA antibody diluent), incubated at room
60min；
(5) PBS rinses 3 times, removes two and resists, and adds DAPI dyeing more than 15min；
(6) rinse 3 times with the PBS of the Tween-20 containing volumn concentration 0.1%, each 10min；
(7) at fluorescence microscopy Microscopic observation.
The Keratin 18 coloration result of galactophore epithelial cell is as shown in Figure 3.
In Fig. 3, A is that the DAPI core of galactophore epithelial cell contaminates to position cell, and in figure, blue circle and ellipse are
Individual cells core (400 ×).B is the skelemin-Keratin 18 immunofluorescence dyeing result of galactophore epithelial cell,
Green represents cellular antigens-antibody response and is positive, and the galactophore epithelial cell of separation expresses Keratin 18 (400 ×).
C is the overlay chart of A and B, shows that detected cell is Keratin 18 positive cell.
Keratin 18 is the skelemin that galactophore epithelial cell is specific expressed, and result above shows, uses immunity glimmering
After light method keratin 18 carries out immunofluorescence dyeing, it was observed that green positive reaction occurs around core, angle egg is described
White 18 are distributed in outside nucleus.Blue portion is the nucleus of DAPI dyeing, and ovalize positions for cell.
In conjunction with cell morphological characteristic, it was demonstrated that cultivating the cell with gained after purification is galactophore epithelial cell.
Three, cell milk composition related gene expression CHARACTERISTICS IDENTIFICATION
The main component in Ruzhong: lactoprotein, lactose and butterfat, be that cell obtains precursor substance, at mammary gland from blood
In epithelial cell after synthesis, it is discharged into mammary gland alveolus.
Selecting milk protein gene CSN1S1, CSN2, LGB, housekeeping gene GAPDH, according to the gene sequence of GenBank
Row, design said gene specific primer, expanded by RT-PCR, analyze cultured cell in vitro secretion characteristic and
The expression of genes of interest, step is as follows:
The galactophore epithelial cell reaching for 5 generations is cultivated after being paved with plate to it, use TRizol cell lysis, extract total
RNA, reverse transcription is cDNA, uses TaKaRa TaqTM(purchased from TaKaRa, catalog number is Version 2.0
R004A) expression of RT-PCR detection milk composition related gene is carried out.
The gene of RT-PCR detection and the primer of correspondence are as shown in table 1.
RT-PCR reaction system and program are the most as shown in tables 2 and 3.
Table 1 RT-PCR amplifying specific primer
Table 2 RT-PCR reaction system
Table 3 RT-PCR program
In galactophore epithelial cell, the RT-PCR testing result of milk composition related gene is as shown in Figure 4.
In Fig. 4, M is DNA marker.
Fig. 4 shows, all has expression, at identical amplification condition in the cell that CSN1S1, CSN2, LGB cultivate in vitro
Under, the expression of CSN2 is relatively low, and the expression of CSN1S1 is higher, illustrates that the cell of in vitro culture has lactation
Ability.
Claims (4)
1. the method separated from cow's milk juice and cultivate galactophore epithelial cell, comprises the steps: to select somatic number in cow's milk juice to be less than 5 × 104Individual/ml and be in the ox of lactation middle and later periods, gathers cow's milk juice；Separate and cultivate the galactophore epithelial cell in cow's milk juice；
Described separation is also cultivated the galactophore epithelial cell in cow's milk juice and is included separating and cultivating primary bovine mammary epithelial cell, more primary bovine mammary epithelial cell is carried out Secondary Culture, obtains the bovine mammary epithelial cell passed on；
The culture medium that described primary bovine mammary epithelial cell and the bovine mammary epithelial cell that passes on use when cultivating is prepared as follows: be 9:1 mixing by DMEM/DF12 culture medium and FBS according to volume ratio, add final concentration of 1 × penicillin, final concentration of 1 × streptomysin, the insulin of final concentration of 5ug/mL, the transferrins of final concentration of 5ug/mL, the hydrocortisone of final concentration of 5ug/mL, the EGF of final concentration of 10ng/mL, the progesterone of final concentration of 1ug/mL, the recombinant bovine galactagogin of final concentration of 10ng/mL；
Described DMEM/DF12 culture medium is purchased from Gibco, and catalog number is C11330500BT；
Described final concentration of 1 × penicillin and final concentration of 1 × the mother liquor of streptomysin be concentration be 100 × the dual anti-solution of penicillin/streptomycin；
Described concentration is 100 × the dual anti-solution of the trade name penicillin/streptomycin (100 ×) of the dual anti-solution of penicillin/streptomycin, purchased from Gibco, catalog number is 15140-122；
Described recombinant bovine galactagogin is purchased from Prospec, and catalog number is CYT-511.
Method the most according to claim 1, it is characterised in that: described ox is milk cow.
Method the most according to claim 2, it is characterised in that: described milk cow is Holstein cow.
4. a culture medium, it is prepared as follows: be 9:1 mixing by DMEM/DF12 culture medium and FBS according to volume ratio, add final concentration of 1 × penicillin, final concentration of 1 × streptomysin, the insulin of final concentration of 5ug/mL, the transferrins of final concentration of 5ug/mL, the hydrocortisone of final concentration of 5ug/mL, the EGF of final concentration of 10ng/mL, the progesterone of final concentration of 1ug/mL, the recombinant bovine galactagogin of final concentration of 10ng/mL；
Described DMEM/DF12 culture medium is purchased from Gibco, and catalog number is C11330500BT；
Described final concentration of 1 × penicillin and final concentration of 1 × the mother liquor of streptomysin be concentration be 100 × the dual anti-solution of penicillin/streptomycin；
Described concentration is 100 × the dual anti-solution of the trade name penicillin/streptomycin (100 ×) of the dual anti-solution of penicillin/streptomycin, purchased from Gibco, catalog number is 15140-122；
Described recombinant bovine galactagogin is purchased from Prospec, and catalog number is CYT-511.
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| CN106811440B (en) * | 2017-02-09 | 2020-09-15 | 广西壮族自治区水牛研究所 | Method for separating and culturing buffalo mammary epithelial cells from buffalo milk |
| CN109837299A (en) * | 2017-11-28 | 2019-06-04 | 中国农业大学 | The method for knocking out galactophore epithelial cell NLRP3 inflammatory body adaptor protein ASC gene |
| CN114317399B (en) * | 2021-08-18 | 2024-04-05 | 川北医学院 | Thyroid organoid culture medium, thyroid organoid culture and passage method |
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Non-Patent Citations (3)
| Title |
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| In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow;Han Hu等;《PLoS One.》;20091103;第4卷(第11期);材料部分和组织分离部分 * |
| 奶牛乳腺上皮细胞体外培养体系的建立;崔立莉 等;《新疆农业科学》;20061231;第43卷(第5期);摘要、第1.3.2节 * |
| 牛乳腺上皮细胞体外培养体系研究进展;宋亚男 等;《生命科学仪器》;20080930;第3.4节 * |
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