CN104059879A - Novel method for culturing chicken macrophages HD11 - Google Patents
Novel method for culturing chicken macrophages HD11 Download PDFInfo
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- CN104059879A CN104059879A CN201410317770.9A CN201410317770A CN104059879A CN 104059879 A CN104059879 A CN 104059879A CN 201410317770 A CN201410317770 A CN 201410317770A CN 104059879 A CN104059879 A CN 104059879A
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Abstract
The invention provides a novel method for culturing chicken HD11 macrophages. Fetal calf serums and chicken serums of different proportions are added to a culture medium RPMI1640 and a culture medium DMEM and cultured for 5 hours, 10 hours, 24 hours and 48 hours respectively, an optimum culture system is detected through an MTT colorimetric test, an optimum culture medium scheme for culturing the chicken macrophages HD11 is acquired, and it shows that the culture effect of the culture medium RPMI1640 is better than that of the culture medium DMEM and the culture effect is optimum when the culture media contain 2.5% of chicken serums.
Description
Technical field
The present invention relates to animal cell culture field and be specifically related to cell culture technology.
Background technology
Scavenger cell belongs to immunocyte, has several functions, is the important object of research cytophagy, cellular immunization and molecular immunology.Chicken HD11 scavenger cell belongs to suspension cell, the existing culture system of this cell: RPMI1640 (Sigma company), 10% new-born calf serum is supplemented (hot deactivation), 10.0mM HEPES, 1.0mM Sodium.alpha.-ketopropionate, 0.1mM non-essential amino acid, 2.0mM glutamine, the penicillin 100 μ g/ml Streptomycin sulphates of 100UmL-1,5 * 10-5M2-mercaptoethanol (pH value 7.3) and 5% carbonic acid gas.The degree of depth of culturing bottle traverse substratum during as incubator surpasses 5mm, and when culturing bottle is uprightly placed, substratum height surpasses 10cm, also needs deep layer to pass into CO2 and air, to guarantee enough gaseous interchange.
The problems such as the current culture system of chicken HD11 scavenger cell exists Growth of Cells slow, and the low and transfection efficiency of proliferate efficiency is low, have affected the application of scavenger cell in related science research, have hindered poultry immunity progress.
Summary of the invention
In order to solve the problem of prior art, it is research object that chicken scavenger cell (HD11 cell) is take in the present invention, improve culture system, with RPMI1640 and two kinds of different culture medias of DMEM, cultivate, and add respectively different ratios foetal calf serum and chicken serum, between different incubation periods, compare HD11 cell cultures effect, be intended to set up a kind of new cultural method.The technical solution used in the present invention is as follows:
A kind of novel method of cultivating chicken HD11 scavenger cell, use respectively two kinds of different substratum of RPMI1640 and DMEM, add foetal calf serum and the chicken serum of different ratios, cultivate respectively after 5 hours, 10 hours, 24 hours, 48 hours, by MTT colorimetric test, recording light absorbs result; Finally take the time as transverse axis, and absorbance value (A) is drawn cell growth curve for the longitudinal axis, detects best culture system, and concrete steps are:
(1) preparation of HD11 cell: get growth conditions good, two bottles, the cell in logarithmic phase growth;
(2) preparation of MTT solution: take 125mg MTT, put into small beaker, add 25mL PBS (0.01mol/L, pH7.4), on magnetic stirrer, stir 30min, strainer filtration sterilization with 0.22 μ m, packing, 4 ℃ of preservations, effective in two weeks, dimethyl sulfoxide (DMSO) (DMSO is used analytical pure product);
(3) substratum, the preparation of foetal calf serum and chicken serum: prepare respectively two kinds of substratum that do not add any serum---RPMI1640 and DMEM, each 50mL, preheating; Each 25mL of preheated foetal calf serum and chicken serum;
The concrete steps of described step (3) are:
1) get growth conditions good, for two bottles of HD11 cells of logarithmic phase growth, counting respectively;
2) collecting cell, cleans 2-3 time with PBS, and centrifugal (1000rpm, 5min) abandons supernatant, resuspended standby with the RPMI1640 and the DMEM that do not add any serum respectively;
3) prepare respectively two kinds of substratum containing Different Chicken serum:
A.RPMI1640
I.RPMI1640+10%FBS+2.5% chicken serum+re-suspended cell;
J.RPMI1640+10%FBS+5% chicken serum+re-suspended cell;
K.RPMI1640+10%FBS+10% chicken serum+re-suspended cell;
L.RPMI1640+15% chicken serum+re-suspended cell;
B.DMEM；
M.DMEM+10%FBS+2.5% chicken serum+re-suspended cell;
N.DMEM+10%FBS+5% chicken serum+re-suspended cell;
O.DMEM+10%FBS+10% chicken serum+re-suspended cell;
P.DMEM+15% chicken serum+re-suspended cell;
4) inoculating cell: by above system, cell is inoculated in 96 orifice plates, and 4 repetitions of each level;
5) cell cultures: put into CO
2incubator, at 5%CO
2, under 37 ℃ of conditions, by the time of design, cultivate respectively 5 hours, 10 hours, 24 hours, 48 hours;
6) colour generation: press after design time cultivation, every hole adds MTT solution (5mg/mL) 20uL, and 37 ℃ are continued to hatch 4 hours, stop cultivating, centrifugal (1000r/min, 15min) carefully discards nutrient solution in hole, every hole adds 150uL DMSO, vibration 10min, and Shi formazan is fully dissolved;
7) colorimetric: select 490nm wavelength, measure each hole absorbance value on enzyme-linked immunosorbent assay instrument, record result.Take the time as transverse axis, and absorbance value (A) is longitudinal axis drafting cell growth curve;
8) result: RPMI1640 substratum and the poor heteropole of DMEM substratum are remarkable; Between different incubation times, comparing difference is not remarkable; Between cultivating with Different Chicken serum-concentration, poor heteropole is remarkable, between two kinds of substratum and different incubation times, makes comparisons mutually, and difference is not remarkable; Between different culture media and Different Chicken serum-concentration, make comparisons mutually, difference is extremely remarkable; The difference of making comparisons mutually between different incubation times and Different Chicken serum-concentration is not remarkable; Three does mutually, and difference is not remarkable; By two kinds of multiple comparisons methods, show, when Different Chicken serum-concentration is cultivated, serum-concentration is 2.5% to have extremely significant difference with other three kinds of chicken serum concentration; And the best medium scheme that obtains cultivating chicken scavenger cell HD11 is: RPMI1640 (Sigma company), and 10% new-born calf serum is supplemented (hot deactivation), 10.0mM HEPES, 1.0mM Sodium.alpha.-ketopropionate, 0.1mM non-essential amino acid, 2.0mM glutamine, 100UmL
-1penicillin 100 μ g/ml Streptomycin sulphates, 5 * 10
-5m2-mercaptoethanol (pH value 7.3), 2.5% chicken serum.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is:
Chicken scavenger cell HD11 can be sooner, better growth, improves proliferate efficiency, and new culture system, increases cell rapidly, and cell viability increases, and shortens test period, and follow-up test is better carried out.
Accompanying drawing explanation
Fig. 1: 5h, DMEM+10% foetal calf serum+2.5% chicken serum;
Fig. 2: 10h, DMEM+10% foetal calf serum+2.5% chicken serum;
Fig. 3: 24h, DMEM+10% foetal calf serum+2.5% chicken serum;
Fig. 4: 48h, DMEM+10% foetal calf serum+2.5% chicken serum;
Fig. 5: 5h, RPMI1640+10% foetal calf serum+2.5% chicken serum;
Fig. 6: 10h, RPMI1640+10% foetal calf serum+2.5% chicken serum;
Fig. 7: 24h, RPMI1640+10% foetal calf serum+2.5% chicken serum;
Fig. 8: 48h, RPMI1640+10% foetal calf serum+2.5% chicken serum;
Growth of Cells figure between Fig. 9 different culture media
Growth of Cells figure between Figure 10 Different Chicken serum-concentration.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail:
Embodiment
Technical scheme of the present invention is: use respectively two kinds of different substratum of RPMI1640 and DMEM, add foetal calf serum and the chicken serum of different ratios, cultivate respectively after 5 hours, 10 hours, 24 hours, 48 hours, by MTT colorimetric test, detect best culture system.
Concrete steps are: first with two kinds of different culture medias, press the time culturing cell arranging; Secondly with MTT colorimetric test, detect, recording light absorbs result; Finally take the time as transverse axis, and absorbance value (A) is longitudinal axis drafting cell growth curve.
1 materials and methods
1.1 material
1.1.1HD11 the preparation of cell
Get growth conditions good, two bottles, the cell in logarithmic phase growth.
1.1.2MTT the preparation of solution
Take 125mg MTT, put into small beaker, add 25mL PBS (0.01mol/L, pH7.4), on magnetic stirrer, stir 30min, with the strainer filtration sterilization of 0.22 μ m, packing, 4 ℃ of preservations.Effective in two weeks.Dimethyl sulfoxide (DMSO) (DMSO is used analytical pure product)
1.1.3 substratum, the preparation of foetal calf serum and chicken serum
Prepare respectively two kinds of substratum that do not add any serum---RPMI1640 and DMEM, each 50mL, preheating; Each 25mL of preheated foetal calf serum and chicken serum.
Concrete steps:
(1) get growth conditions good, for two bottles of HD11 cells of logarithmic phase growth, counting respectively;
(2) collecting cell, cleans 2-3 time with PBS, and centrifugal (1000rpm, 5min) abandons supernatant, resuspended standby with the RPMI1640 and the DMEM that do not add any serum respectively;
(3) prepare respectively two kinds of substratum containing Different Chicken serum:
A.RPMI1640
Q.RPMI1640+10%FBS+2.5% chicken serum+re-suspended cell
R.RPMI1640+10%FBS+5% chicken serum+re-suspended cell
S.RPMI1640+10%FBS+10% chicken serum+re-suspended cell
T.RPMI1640+15% chicken serum+re-suspended cell
B.DMEM
U.DMEM+10%FBS+2.5% chicken serum+re-suspended cell
V.DMEM+10%FBS+5% chicken serum+re-suspended cell
W.DMEM+10%FBS+10% chicken serum+re-suspended cell
X.DMEM+15% chicken serum+re-suspended cell
(4) inoculating cell: by above system, cell is inoculated in 96 orifice plates, and 4 repetitions of each level.
(5) cell cultures: put into CO
2incubator, at 5%CO
2, under 37 ℃ of conditions, by the time of design, cultivate respectively 5 hours, 10 hours, 24 hours, 48 hours.
(6) colour generation: press after design time cultivation, every hole adds MTT solution (5mg/mL) 20uL, and 37 ℃ are continued to hatch 4 hours, stop cultivating, centrifugal (1000r/min, 15min) carefully discards nutrient solution in hole, every hole adds 150uL DMSO, vibration 10min, and Shi formazan is fully dissolved.
(7) colorimetric: select 490nm wavelength, measure each hole absorbance value on enzyme-linked immunosorbent assay instrument, record result.Take the time as transverse axis, and absorbance value (A) is longitudinal axis drafting cell growth curve.
2 results
Culture effect comparison between 2.1 different culture systems: in Table 1
Table 1: culture effect comparison between different culture systems
Note: when same column compares, " * * " represent that difference is extremely remarkable, " * " expression significant difference; When colleague compares, capitalization " A " or " B " or " C " represent that difference is extremely remarkable, lowercase " a " or " b " or " c " or " d " expression significant difference.
Variance analysis between 2.2 different incubation times, substratum, different serum-concentration
Variance analysis between the different incubation times of table 2, substratum, different serum-concentration
| Source | III type sum of squares | df | All square | F | Sig. |
| Calibration model | 1.996 a | 31 | .064 | 5.910 | .000 |
| Intercept | 1.044 | 1 | 1.044 | 95.810 | .000 |
| medium | 1.044 | 1 | 1.044 | 95.787 | .000 |
| time | .026 | 3 | .009 | .802 | .498 |
| serum | .272 | 3 | .091 | 8.317 | .000 |
| medium*time | .026 | 3 | .009 | .803 | .497 |
| medium*serum | .272 | 3 | .091 | 8.317 | .000 |
| time*serum | .178 | 9 | .020 | 1.817 | .082 |
| medium*time*serum | .178 | 9 | .020 | 1.817 | .082 |
| Error | .697 | 64 | .011 |
| Amount to | 3.737 | 96 | |||
| The total of proofreading and correct | 2.693 | 95 |
By upper table data, draw, RPMI1640 substratum and the poor heteropole of DMEM substratum are remarkable; Between different incubation times, comparing difference is not remarkable; Between cultivating with Different Chicken serum-concentration, poor heteropole is remarkable.Between two kinds of substratum and different incubation times, make comparisons mutually, difference is not remarkable; Between different culture media and Different Chicken serum-concentration, make comparisons mutually, difference is extremely remarkable; The difference of making comparisons mutually between different incubation times and Different Chicken serum-concentration is not remarkable; Three does mutually, and difference is not remarkable.
Multiple comparisons between 2.3 Different Chicken serum-concentrations:
Multiple comparisons between table 3 Different Chicken serum-concentration
By upper table, drawn: by two kinds of multiple comparisons methods, show, when Different Chicken serum-concentration is cultivated, serum-concentration is 2.5% to have extremely significant difference with other three kinds of chicken serum concentration.
2.4 Growth of Cells figure
2.4.1 the Growth of Cells comparison (Fig. 9) between different culture media
Scheme thus knownly, the culture effect of substratum RPMI1640 is better than with substratum DMEM and cultivates.
2.4.2 the Growth of Cells figure (Figure 10) between Different Chicken serum-concentration
When substratum contains 2.5% chicken serum, culture effect is optimum as seen from the figure.
Concrete cell cultures is shown in accompanying drawing 1-8.
3. conclusion
By above data, shown, the best medium scheme of cultivating chicken scavenger cell HD11 is: RPMI1640 (Sigma company), 10% new-born calf serum is supplemented (hot deactivation), 10.0mM HEPES, 1.0mM Sodium.alpha.-ketopropionate, 0.1mM non-essential amino acid, 2.0mM glutamine, the penicillin 100 μ g/ml Streptomycin sulphates of 100UmL-1,5 * 10-5M2-mercaptoethanol (pH value 7.3), 2.5% chicken serum.
Claims (3)
1. a novel method of cultivating chicken HD11 scavenger cell, it is characterized in that: use respectively two kinds of different substratum of RPMI1640 and DMEM, add foetal calf serum and the chicken serum of different ratios, cultivate respectively after 5 hours, 10 hours, 24 hours, 48 hours, by MTT colorimetric test, recording light absorbs result; Finally take the time as transverse axis, and absorbance value (A) is drawn cell growth curve for the longitudinal axis, detects best culture system, and concrete steps are:
(1) preparation of HD11 cell: get growth conditions good, two bottles, the cell in logarithmic phase growth;
(2) preparation of MTT solution: take 125mg MTT, put into small beaker, add 25mL PBS (0.01mol/L, pH7.4), on magnetic stirrer, stir 30min, strainer filtration sterilization with 0.22 μ m, packing, 4 ℃ of preservations, effective in two weeks, dimethyl sulfoxide (DMSO) (DMSO is used analytical pure product);
(3) substratum, the preparation of foetal calf serum and chicken serum: prepare respectively two kinds of substratum that do not add any serum---RPMI1640 and DMEM, each 50mL, preheating; Each 25mL of preheated foetal calf serum and chicken serum;
The concrete steps of described step (3) are:
1) get growth conditions good, for two bottles of HD11 cells of logarithmic phase growth, counting respectively;
2) collecting cell, cleans 2-3 time with PBS, and centrifugal (1000rpm, 5min) abandons supernatant, resuspended standby with the RPMI1640 and the DMEM that do not add any serum respectively;
3) prepare respectively two kinds of substratum containing Different Chicken serum:
A.RPMI1640
A.RPMI1640+10%FBS+2.5% chicken serum+re-suspended cell;
B.RPMI1640+10%FBS+5% chicken serum+re-suspended cell;
C.RPMI1640+10%FBS+10% chicken serum+re-suspended cell;
D.RPMI1640+15% chicken serum+re-suspended cell;
B.DMEM；
E.DMEM+10%FBS+2.5% chicken serum+re-suspended cell;
F.DMEM+10%FBS+5% chicken serum+re-suspended cell;
G.DMEM+10%FBS+10% chicken serum+re-suspended cell;
H.DMEM+15% chicken serum+re-suspended cell;
4) inoculating cell: by above system, cell is inoculated in 96 orifice plates, and 4 repetitions of each level;
5) cell cultures: put into CO
2incubator, at 5%CO
2, under 37 ℃ of conditions, by the time of design, cultivate respectively 5 hours, 10 hours, 24 hours, 48 hours;
6) colour generation: press after design time cultivation, every hole adds MTT solution (5mg/mL) 20uL, and 37 ℃ are continued to hatch 4 hours, stop cultivating, centrifugal (1000r/min, 15min) carefully discards nutrient solution in hole, every hole adds 150uL DMSO, vibration 10min, and Shi formazan is fully dissolved;
7) colorimetric: select 490nm wavelength, measure each hole absorbance value on enzyme-linked immunosorbent assay instrument, record result.Take the time as transverse axis, and absorbance value (A) is longitudinal axis drafting cell growth curve;
8) result: RPMI1640 substratum and the poor heteropole of DMEM substratum are remarkable; Between different incubation times, comparing difference is not remarkable; Between cultivating with Different Chicken serum-concentration, poor heteropole is remarkable, between two kinds of substratum and different incubation times, makes comparisons mutually, and difference is not remarkable; Between different culture media and Different Chicken serum-concentration, make comparisons mutually, difference is extremely remarkable; The difference of making comparisons mutually between different incubation times and Different Chicken serum-concentration is not remarkable; Three does mutually, and difference is not remarkable; By two kinds of multiple comparisons methods, show, when Different Chicken serum-concentration is cultivated, serum-concentration is 2.5% to have extremely significant difference with other three kinds of chicken serum concentration; And the best medium scheme that obtains cultivating chicken scavenger cell HD11 is: RPMI1640 (Sigma company), and 10% new-born calf serum is supplemented (hot deactivation), 10.0mM HEPES, 1.0mM Sodium.alpha.-ketopropionate, 0.1mM non-essential amino acid, 2.0mM glutamine, 100UmL
-1penicillin 100 μ g/ml Streptomycin sulphates, 5 * 10
-5m2-mercaptoethanol (pH value 7.3), 2.5% chicken serum.
2. a novel method for cultivation chicken HD11 scavenger cell according to claim 1, is characterized in that: the culture effect of substratum RPMI1640 is better than with substratum DMEM and cultivates.
3. a novel method for cultivation chicken HD11 scavenger cell according to claim 1, is characterized in that: when substratum contains 2.5% chicken serum, culture effect is optimum.
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|---|---|---|---|
| CN201410317770.9A CN104059879A (en) | 2014-07-04 | 2014-07-04 | Novel method for culturing chicken macrophages HD11 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779392A (en) * | 2016-05-20 | 2016-07-20 | 广州赛莱拉干细胞科技股份有限公司 | Macrophage culture medium and culture method |
| CN114292809A (en) * | 2021-12-31 | 2022-04-08 | 吉林国健生命工程科学技术有限公司 | Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness |
| CN114634907A (en) * | 2022-04-01 | 2022-06-17 | 香港大学深圳医院 | System and method for co-culturing macrophage and liver cancer cell |
-
2014
- 2014-07-04 CN CN201410317770.9A patent/CN104059879A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| BERND KASPERS 等: "Chicken macrophages and thrombocytes share a common cell surface antigen defined by a monoclonal antibody", 《VET IMMUNOL IMMUNOPATHOL.》 * |
| IRENA OVEN等: "Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses", 《VET RES.》 * |
| 郭涛 等: "不同培养基和不同体积分数小牛血清对许旺细胞生长状态的影响", 《中国组织工程研究与临床康复》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779392A (en) * | 2016-05-20 | 2016-07-20 | 广州赛莱拉干细胞科技股份有限公司 | Macrophage culture medium and culture method |
| CN114292809A (en) * | 2021-12-31 | 2022-04-08 | 吉林国健生命工程科学技术有限公司 | Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness |
| CN114634907A (en) * | 2022-04-01 | 2022-06-17 | 香港大学深圳医院 | System and method for co-culturing macrophage and liver cancer cell |
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