CN1632576A - Dedicated Hemolysin for flow cytometer and preparing method and application thereof - Google Patents
Dedicated Hemolysin for flow cytometer and preparing method and application thereof Download PDFInfo
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- CN1632576A CN1632576A CN 200510042001 CN200510042001A CN1632576A CN 1632576 A CN1632576 A CN 1632576A CN 200510042001 CN200510042001 CN 200510042001 CN 200510042001 A CN200510042001 A CN 200510042001A CN 1632576 A CN1632576 A CN 1632576A
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- hemolysin
- flow cytometer
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Abstract
It is a process method of hemolysin in special cell device and its application, which belongs to medical test agent technique field. The hemolysin is composed of NaCI, Na#-[2] HPO#-[4].12H#-[2] O, KH#-[2] PO#-[4], cavaform and double stilled water. Each component is mixed and is put in the water for four to five hours and is shaken every thirty to forty minutes and to be filtered to get the agent. The hemolysin in this invention is used as surface marks of leucocyte in blood tested by the flow cell device.
Description
(1) technical field
Used hemolysin and preparation method thereof and application when the present invention relates to flow cytometer detection each group of blood leucocyte (lymphocyte, monocyte, granulocyte) surface marker.
(2) background technology
Flow cytometer has been applied to the every field of scientific research and clinical medicine check, and as the inspection of immunologic function, tumour antigen, metastatic gene and tumor suppressor gene and other disease related genes detect, Apoptosis, AIDS diagnosis and prognosis judgement etc.Along with popularizing and application of flow cytometer, the exploitation of its matched reagent and development also seem more and more important.At present, the used hemolysin of flow cytometer relies on import substantially, mainly contains the product of U.S. Becton Dickinson (BD) company and Beckman Coulter company, and it costs an arm and a leg, and can not in time supply, and brings very big inconvenience and waste to the user.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, dedicated Hemolysin for flow cytometer and preparation method thereof and application are provided.
Dedicated Hemolysin for flow cytometer of the present invention composed as follows:
NaCl????????????????5～12g
Na
2HPO
4·12H
2O??1～8g
Or Na
2HPO
4.2H
2O 0.5～6g
Or K
2HPO
45～10g
KH
2PO
4???????????0.1～0.6g
Or NaH
2PO
4.2H
2O 0.1～3g
KCl?????????????????0～0.5g
Paraformaldehyde [(CH
2O) n] 20～50g
Add distilled water to 1L
Paraformaldehyde molecular formula: (CH
2O) n, wherein n=8-100.
The preparation method of dedicated Hemolysin for flow cytometer of the present invention is as follows:
1. raw material weighing: with required various raw materials by the weighing respectively of above formula rate.
2. preparation: will be various load weighted raw materials mixing, put in the 45-50 ℃ of water-bath 4-5 hour, therebetween once every 30-40 minute stirring and evenly mixing.
3. filter: above-mentioned solution is removed precipitation with double-deck filter paper filtering, promptly get hemolysin of the present invention.
Carry out the product packing then: will filter good product and carry out packing, the 50ml/ bottle.The product that branch is installed posts label, mark operation instruction and points for attention etc.Finished product acceptance(check) warehouse-in.
Dedicated Hemolysin for flow cytometer of the present invention is used for flow cytometer and detects each group of blood leucocyte (lymphocyte, monocyte, granulocyte) surface marker.Specifically can be used for each serial flow cytometer that BD, Beckman Coulter or other company produce.
Concrete using method is as follows:
Method one:
1. by peripheral blood: hemolysin is that 1: 1 volume ratio joins hemolysin in the anticoagulation cirumferential blood, low speed mixing, room temperature 10 minutes.
2. the distilled water that adds 20 times of volumes, mixing room temperature 5-10 minute, dissolves red blood cell fully.Flow cytometer detects each group of blood leucocyte (lymphocyte, monocyte, granulocyte) surface marker.
Method two:
1. use preceding with hemolysin 10 times of dilutions of distilled water, mixing.
2. will dilute hemolysin by 20: 1 volume ratios and join in the anticoagulation cirumferential blood, the low speed mixing room temperature 5-10 minute, dissolves red blood cell fully.Flow cytometer detects each group of blood leucocyte (lymphocyte, monocyte, granulocyte) surface marker.
Excellent results compared with prior art of the present invention is as follows:
1. comprise paraformaldehyde in the dedicated Hemolysin for flow cytometer component of the present invention, paraformaldehyde has fixedly leucocyte membrane interaction, can farthest protect the leucocyte film complete, be not damaged, and keep the leucocyte membranous antigen unaffected.Paraformaldehyde has fixation to pathogenic microorganism such as hepatitis B, the SARS-CoV etc. that may exist in the blood, reduces its toxicity and infectiousness.Avoid the infection of causal organism with protection environment and experiment operator.
2. the preferred pH value 5.0-8.0 of hemolysin if pH value is too low or too high, increases leukocytic destruction.Comprise phosphate buffer in the dedicated Hemolysin for flow cytometer component of the present invention, pH value is 7.2-7.4, and is comparatively stable, and this pH value is the most suitable pH value of leucocyte existence near the pH value of blood of human body, can protect leucocyte to avoid destroying and the antigen change.
3. contain a large amount of water constituents in the dedicated Hemolysin for flow cytometer working concentration of the present invention, this is to make haemoclastic principal ingredient.Water relies on hypotonic effect, red blood cell is risen split, and reaches haemoclastic effect.
4. each composition of dedicated Hemolysin for flow cytometer of the present invention is cheap, and supply is convenient, obtains easily, and cost of products and cost are reduced.
5. dedicated Hemolysin for flow cytometer manufacture craft of the present invention is simple, and layoutprocedure does not need expensive instrument and equipment condition, makes production simple.
6. dedicated Hemolysin for flow cytometer using method of the present invention is simple and easy, variation, and the user can select voluntarily according to the custom of oneself, and is convenient and easy.
7. dedicated Hemolysin for flow cytometer of the present invention does not have toxicity, and user and environment are not had harm.
(4) description of drawings
The leucocyte forward scattering light (FSC) that Fig. 1 is to use the hemolysin of the embodiment of the invention 1 to act on to obtain on U.S. company BD FACSCalibur flow cytometer behind people's anticoagulation cirumferential blood and the dot-density plot of side scattered light (SSC), visible leucocyte are divided into three groups in lymphocyte, monocyte, granulocyte.
Fig. 2 is that hemolysin that BD company produces acts on the leucocyte forward scattering light (FSC) that obtains behind people's anticoagulation cirumferential blood and the dot-density plot of side scattered light (SSC) on U.S. company BD FACSCalibur flow cytometer.As seen leucocyte is divided into three groups in lymphocyte, monocyte, granulocyte.Also have significantly cell fragment in addition, this is owing to erythrocytic dissolving does not thoroughly cause.
Fig. 3,4 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the lymphocyte CD 14 expression histograms that obtain behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.
Fig. 5,6 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the monocyte CD14 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.
Fig. 7,8 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the granulocyte CD14 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.
Fig. 9,10 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the lymphocyte CD 45 expression histograms that obtain behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.
Figure 11,12 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the monocyte CD45 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.
Figure 13,14 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the granulocyte CD45 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.
(5) embodiment
Embodiment 1: dedicated Hemolysin for flow cytometer composed as follows:
NaCl????????????????8g
Na
2HPO
4.12H
2O???2.89g
KH
2PO
4???????????0.2g
KCl?????????????????0.2g
Paraformaldehyde 30g
Add distilled water to 1L
The preparation method:
1. raw material weighing: with required various raw materials by the weighing respectively of above formula rate.
2. preparation: various load weighted raw materials are mixed, put into 50 ℃ of water-baths 4 hours, every 30 minutes stirring and evenly mixings once therebetween.
3. filter: double-deck filter paper filtering is removed precipitation.
Carry out the product packing then: will filter good product and carry out packing, the 50ml/ bottle.The product that branch is installed posts label, mark operation instruction and points for attention etc.Finished product acceptance(check) warehouse-in.
Table 1 is lymphocyte, monocyte, granulocyte CD14, the CD45 expression statistics that 40 routine people's anticoagulation cirumferential bloods obtain on U.S. company BD FACSCalibur flow cytometer under the hemolysin of dedicated Hemolysin for flow cytometer embodiment 1 of the present invention and the production of BD company acts on respectively, and it is straightforward that the two relatively has height one.This shows that using dedicated Hemolysin for flow cytometer of the present invention to detect leucocyte (lymphocyte, monocyte, granulocyte) membranous antigen expression and the hemolysin that uses BD company to produce detects leucocyte (lymphocyte, monocyte, granulocyte) membranous antigen expression result and have good one straightforward.
Table 1. uses the hemolysin and the BD hemolysin of the embodiment of the invention 1
Lymphocyte, monocyte, granulocyte CD14, CD45 expression of results
| The example number | CD14 expresses (%) | CD45 expresses (%) | |||
| Hemolysin of the present invention | The BD hemolysin | Hemolysin of the present invention | The BD hemolysin | ||
| Lymphocyte | ????40 | ???1.01±0.24 | ?1.00±0.23 | ?98.94±0.71 | ??98.90±0.72 |
| Monocyte | ????40 | ???92.80±1.98 | ?92.81±1.94 | ?95.15±2.73 | ??95.11±2.76 |
| Granulocyte | ????40 | ???91.16±3.68 | ?91.22±3.76 | ?99.39±0.48 | ??99.41±0.47 |
The concrete using method of present embodiment hemolysin is: by peripheral blood: hemolysin is that 1: 1 volume ratio joins hemolysin in the anticoagulation cirumferential blood, low speed mixing, room temperature 10 minutes.Add the distilled water of 20 times of volumes, mixing room temperature 5-10 minute, dissolves red blood cell fully.Detect each group of blood leucocyte (lymphocyte, monocyte, granulocyte) surface marker with flow cytometer.
The leucocyte forward scattering light (FSC) that Fig. 1 is to use the hemolysin of the embodiment of the invention 1 to act on to obtain on U.S. company BD FACSCalibur flow cytometer behind people's anticoagulation cirumferential blood and the dot-density plot of side scattered light (SSC), visible leucocyte are divided into three groups in lymphocyte, monocyte, granulocyte.
Fig. 2 is that hemolysin that BD company produces acts on the leucocyte forward scattering light (FSC) that obtains behind people's anticoagulation cirumferential blood and the dot-density plot of side scattered light (SSC) on U.S. company BD FACSCalibur flow cytometer.As seen leucocyte is divided into three groups in lymphocyte, monocyte, granulocyte.Also have significantly cell fragment in addition, this is owing to erythrocytic dissolving does not thoroughly cause.
Fig. 3,4 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the lymphocyte CD 14 expression histograms that obtain behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.As seen the two CD14 expression has good one straightforward.
Fig. 5,6 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the monocyte CD14 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.As seen the two CD14 expression has good one straightforward.
Fig. 7,8 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the granulocyte CD14 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.As seen the two CD14 expression has good one straightforward.
Fig. 9,10 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the lymphocyte CD 45 expression histograms that obtain behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.As seen the two CD45 expression has good one straightforward.
Figure 11,12 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the monocyte CD45 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.As seen the two CD45 expression has good one straightforward.
Figure 13,14 is respectively the hemolysin of the use embodiment of the invention 1 and the hemolysin of BD company production acts on the granulocyte CD45 expression histogram that obtains behind people's anticoagulation cirumferential blood on U.S. company BD FACSCalibur flow cytometer.As seen the two CD45 expression has good one straightforward.
Embodiment 2.
The preparation method is with embodiment 1, and different is that hemolysin is composed as follows:
NaCl?????????????????7.2g
Na
2HPO
4·12H
2O???3.73g
KH
2PO
4????????????0.43g
Paraformaldehyde 40g
Add distilled water to 1L
Embodiment 3.
The preparation method is with embodiment 1, and different is that hemolysin is composed as follows:
NaCl????????????????9g
Na
2HPO
4·12H
2O??6g
NaH
2PO
4·2H
2O???0.4g
Paraformaldehyde 25g
Add distilled water to 1L
Embodiment 4.
The preparation method is with embodiment 1, and different is that hemolysin is composed as follows:
NaCl???????????????7.2g
K
2HPO
4??????????7.52g
NaH
2PO
4·2H
2O??1.49g
Paraformaldehyde 30g
Add distilled water to 1L
Claims (5)
1. dedicated Hemolysin for flow cytometer is characterized in that, and is composed as follows:
NaCl????????????????????????????????5～12g
Na
2HPO
4·12H
2O??????????????????1～8g
Or Na
2HPO
4.2H
2O 0.5～6g
Or K
2HPO
45～10g
KH
2PO
4???????????????????????????0.1～0.6g
Or NaH
2PO
4.2H
2O 0.1～3g
KCl?????????????????????????????????0～0.5g
Paraformaldehyde [(CH
2O) n], n=8-100 20～50g
Add distilled water to 1L.
2. the preparation method of the described dedicated Hemolysin for flow cytometer of claim 1, step is as follows:
(1) raw material weighing: with the weighing respectively in proportion of required various raw materials;
(2) preparation: will be various load weighted raw materials mixing, put in the 45-50 ℃ of water-bath 4-5 hour, therebetween once every 30-40 minute stirring and evenly mixing;
(3) filter: above-mentioned solution is removed precipitation with double-deck filter paper filtering, promptly get hemolysin.
3. the described dedicated Hemolysin for flow cytometer of claim 1 is used for each group of flow cytometer detection blood leucocyte surface marker.
4. as the purposes of dedicated Hemolysin for flow cytometer as described in the claim 3, it is characterized in that using method is as follows:
By peripheral blood: hemolysin is that 1: 1 volume ratio joins hemolysin in the anticoagulation cirumferential blood, low speed mixing, room temperature 10 minutes; Add the distilled water of 20 times of volumes, mixing room temperature 5-10 minute, dissolves red blood cell fully; Detect with flow cytometer.
5. as the purposes of dedicated Hemolysin for flow cytometer as described in the claim 3, it is characterized in that using method is as follows:
Use preceding with hemolysin 10 times of dilutions of distilled water, mixing; To dilute hemolysin by 20: 1 volume ratios and join in the anticoagulation cirumferential blood, the low speed mixing room temperature 5-10 minute, dissolves red blood cell fully; Detect with flow cytometer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510042001 CN1287151C (en) | 2005-01-04 | 2005-01-04 | Dedicated Hemolysin for flow cytometer and preparing method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510042001 CN1287151C (en) | 2005-01-04 | 2005-01-04 | Dedicated Hemolysin for flow cytometer and preparing method and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1632576A true CN1632576A (en) | 2005-06-29 |
| CN1287151C CN1287151C (en) | 2006-11-29 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Country Status (1)
| Country | Link |
|---|---|
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101322145B (en) * | 2005-10-19 | 2011-11-23 | 沃德劳有限合伙公司 | Apparatus and method for performing counts within a biologicfluid sample |
| CN105136687A (en) * | 2015-09-07 | 2015-12-09 | 合肥金域医学检验所有限公司 | Improved erythrocyte osmotic fragility detection method |
| CN106255886A (en) * | 2014-04-03 | 2016-12-21 | 学校法人东京女子医科大学 | The method of detection survival motor neuron protein expression |
| CN106525699A (en) * | 2016-10-21 | 2017-03-22 | 深圳市职业病防治院 | Peripheral blood lymphocyte micronucleus detection kit and detection method thereof |
| CN109187121A (en) * | 2018-08-06 | 2019-01-11 | 中国人民解放军第三0二医院 | Reagent and its application in combined immunization effect early stage fast appraisement method |
| CN110501490A (en) * | 2019-09-12 | 2019-11-26 | 核工业总医院 | A kind of isotonic hemolysin and preparation method thereof, processing biological sample method and detection leucocyte membranous antigen method |
| JP2020030180A (en) * | 2018-08-24 | 2020-02-27 | 東ソー株式会社 | Hemolytic agent for blood sample |
-
2005
- 2005-01-04 CN CN 200510042001 patent/CN1287151C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101322145B (en) * | 2005-10-19 | 2011-11-23 | 沃德劳有限合伙公司 | Apparatus and method for performing counts within a biologicfluid sample |
| CN106255886A (en) * | 2014-04-03 | 2016-12-21 | 学校法人东京女子医科大学 | The method of detection survival motor neuron protein expression |
| CN105136687A (en) * | 2015-09-07 | 2015-12-09 | 合肥金域医学检验所有限公司 | Improved erythrocyte osmotic fragility detection method |
| CN106525699A (en) * | 2016-10-21 | 2017-03-22 | 深圳市职业病防治院 | Peripheral blood lymphocyte micronucleus detection kit and detection method thereof |
| CN109187121A (en) * | 2018-08-06 | 2019-01-11 | 中国人民解放军第三0二医院 | Reagent and its application in combined immunization effect early stage fast appraisement method |
| JP2020030180A (en) * | 2018-08-24 | 2020-02-27 | 東ソー株式会社 | Hemolytic agent for blood sample |
| JP7143678B2 (en) | 2018-08-24 | 2022-09-29 | 東ソー株式会社 | Methods for detecting target cells |
| CN110501490A (en) * | 2019-09-12 | 2019-11-26 | 核工业总医院 | A kind of isotonic hemolysin and preparation method thereof, processing biological sample method and detection leucocyte membranous antigen method |
| CN110501490B (en) * | 2019-09-12 | 2022-06-10 | 核工业总医院 | Isotonic hemolysin, preparation method thereof, method for treating biological sample and method for detecting leukocyte membrane antigen |
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| Publication number | Publication date |
|---|---|
| CN1287151C (en) | 2006-11-29 |
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