CN112730831A - Application of cell sorting system based on LEPR monoclonal antibody specificity recognition - Google Patents
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Abstract
The invention discloses a sorting system for specifically recognizing cells based on an LEPR monoclonal antibody, which comprises the following components: the kit comprises a LEPR monoclonal antibody, a fluorescent secondary antibody and hair follicle dermal papilla cells, wherein the LEPR monoclonal antibody is used for specifically marking the hair follicle dermal papilla cells, and the fluorescent secondary antibody is used for specifically marking the LEPR monoclonal antibody. The LEPR monoclonal antibody of the present invention can specifically and immunologically bind to DP cells (hair follicle dermal papilla cells) for sorting the DP cells, and the DP cells have the ability to promote hair growth when injected subcutaneously, and can reconstitute hair when mixed with epithelial stem cells.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of a sorting system for dermal papilla cells of mammals based on LEPR monoclonal antibody specificity recognition.
Background
Dermal papilla cells (DP cells) develop from mesoderm, are special mesenchymal stem cells, play an important role in the formation of hair follicles and hair growth, and release signals directly regulate the maintenance, activation, growth and differentiation of hair follicle stem cells and melanin stem cells in hair follicles; in vitro, DP cells and hair follicle stem cells can be mixed to reconstruct hair follicle tissue regenerated hair, mouse experiments prove that the DP cells can be injected into the subcutaneous tissue to induce the hair to enter a growth phase, and the DP cells can secrete various growth factors, so the DP cells can also support the in vitro proliferation and the maintenance of dryness of skin stem cells, hair follicle stem cells and melanin stem cells, and have very important significance for treating related diseases such as alopecia, leukotrichia, leucoderma, burn, trauma, skin genetic diseases and the like by utilizing the DP cells. The existing methods for separating DP cells include the following 3 methods:
1. adopting a micro-surgery dissection mode: DP cells were isolated by physical separation using a dissecting needle, forceps, scissors, and the like.
2. The method using transgenic mice: the DP cells are expressed with a specific fluorescent protein by using a transgenic technology, and then are sorted and collected by using a flow cytometer. For example, LEF1 has been used as a promoter to express fluorescent proteins, while LEF1 is expressed only in DP cells, thereby allowing the sorting of DP cells by sorting fluorescent proteins.
3. Specifically labeling DP cells by using a CD133 antibody coupled with biotin, and screening a fluorescent secondary antibody for recognizing the biotin by using a flow cytometer, thereby sorting the DP cells.
However, methods of microdissection to isolate DP cells are time consuming, labor intensive, inefficient, and only suitable for dissecting relatively large hair follicles, such as mouse beard hair follicles, human scalp hair follicles. For hair follicles of small size, such as those on the skin of the back of a mouse, this is not possible. The DP cell is separated by using the transgenic mouse method, the cost for constructing the transgenic mouse is higher, the operation is complex, and the process of breeding the mouse is longer. The CD133 antibody is used for labeling DP cells of mouse dorsal skin hair follicles, and then the DP cells combined with the fluorescent secondary antibody are sorted by a flow cytometer, so that only one cell membrane protein CD133 can be used for sorting the DP cells at present, but the CD133 cannot specifically recognize the DP cells, as shown in figure 1, and therefore, the DP cells cannot be effectively sorted.
Accordingly, those skilled in the art have endeavored to develop a method capable of specifically recognizing and isolating a large number of DP cells in vitro.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for separating dermal papilla cells of mammals, which is suitable for the micro-dissection of DP cells and the relatively large hair follicles.
In order to achieve the above object, the present invention provides an application of a cell sorting system based on specific recognition of LEPR monoclonal antibody, wherein the cell sorting system comprises the following components: the kit comprises a LEPR monoclonal antibody, a fluorescent secondary antibody and hair follicle dermal papilla cells, wherein the LEPR monoclonal antibody is used for specifically marking the hair follicle dermal papilla cells, and the fluorescent secondary antibody is used for specifically marking the LEPR monoclonal antibody.
Further, the method for sorting the dermal papilla cells of the hair follicle by the cell sorting system comprises the following steps:
(1) preparing the hair follicle dermal papilla cell suspension by adopting an enzyme digestion method,
(2) specifically labeling hair follicle dermal papilla cells in the cell suspension with an LEPR monoclonal antibody,
(3) the fluorescent secondary antibody is specifically combined with the LEPR monoclonal antibody,
(4) and (4) sorting hair follicle dermal papilla cells according to the sorting condition set by cross-gating the emission wavelength of the fluorescent secondary antibody.
Further, the step (1) is specifically the following steps:
(1.1) selecting mice with hair in the growth period, killing the mice by a neck-breaking method,
(1.2) scratching the skin area, shaving off the hair, washing the skin,
(1.3) preparing a 10cm culture dish in advance, putting precooled 1 XPBS buffer solution on ice, cutting skin, putting the skin in the culture dish, washing the 1 XPBS buffer solution,
(1.4) the dermis was faced up, the fat layer on the dermis side was torn off with forceps without damaging the hair follicle,
(1.5) placing 3mL of 0.25% collagenase in a 6cm culture dish, preheating to 37 ℃, placing the skin in collagenase for digestion at 37 ℃, wherein the digestion time is 0.5-2 h, the fully digested skin is transparent,
(1.6) preparing a new 6cm dish, placing 5mL room temperature 1 XPBS in advance, transferring the skin to the new dish, washing off collagenase gently, repeating 2 times,
(1.7) sucking off PBS, adding 3mL of 0.25% pancreatin, placing in a shaking table at 37 ℃, digesting for 20-30min at 25rpm, transferring to ice after digestion, and then operating on ice or in an environment at 4 ℃,
(1.8) cutting the skin into small pieces of 2mm multiplied by 2mm,
(1.9) adding 3mL of DMEM, terminating the pancreatin digestion, pipetting 10-12 times in total volume with a 10mL pipette to form a cell suspension,
(1.10) passing the cell suspension through a 40 μm cell filter, transferring the filtrate to a 50mL centrifuge tube,
(1.11) washing the culture dish with DMEM, passing the washing solution through a 40 μm cell filter, transferring the filtrate to a 50mL centrifuge tube,
(1.12) centrifuging at 4 ℃ for 5min at 1500rpm, sucking off the supernatant,
(1.13) resuspend the cells with pre-cooled PBS-F buffer and count, gently blow well with 2mL PBS-F buffer, then resuspend the hair follicle dermal papilla cell suspension with 3 mL.
Further, the preparation method of the LEPR monoclonal antibody comprises the following steps:
(i) two New Zealand white rabbits are immunized by LEPR recombinant protein antigen, the serum titer is detected by an ELISA method,
(ii) b cell in vitro culture and supernatant screening positive clones aiming at specific antigen,
(iii) a recombinant LEPR monoclonal antibody is disclosed,
(iv) and (3) preparing the large-quality-improved particles of the target clone, then carrying out transfection production, carrying out ProA affinity column purification on the transfection supernatant to obtain the LEPR monoclonal antibody, carrying out buffer solution replacement, and finally storing the LEPR monoclonal antibody in a PBS buffer solution.
Further, in the step (i), the immune part is injected at multiple points under the skin, and the qualified standard of the immune titer is OD1:64000>0.8。
Further, the step (ii) is specifically: taking rabbit spleen, separating B cells, plating, culturing in vitro, adding cell growth factor and feeder cells, culturing for 10-15 days, and detecting supernatant; taking the supernatant, carrying out primary screening, and selecting positive clones aiming at specific antigens; performing WB \ FC \ ICC \ IHC verification on the supernatant of the ELISA positive clone; all positive clones were selected, cell lysates prepared and stored at-80 ℃.
Further, step (iii) is specifically to take Top10 clone for antibody recombination; using specific PCR primers to call variable region sequences of heavy chains and light chains; after sequencing verification, plasmid construction is carried out, and small upgraded particles are prepared for transfection test; after transfection and positive detection of supernatant ELISA, WB \ FC \ ICC \ IHC verification is carried out; and determining the positive clone antibody recombinant plasmid pair.
The invention also provides a kit for sorting hair follicle dermal papilla cells, which is characterized by comprising a LEPR monoclonal antibody, a fluorescent secondary antibody and a PBS buffer solution, wherein the LEPR monoclonal antibody specifically marks the hair follicle dermal papilla cells, and the fluorescent secondary antibody specifically marks the LEPR monoclonal antibody.
In another aspect, the invention provides the use of hair follicle dermal papilla cells specifically recognized based on LEPR monoclonal antibodies in hair growth.
Technical effects
Compared with the prior art, the cell sorting system can specifically and efficiently sort a large number of hair follicle DP cells.
The sorted cells of the invention have functionality after in vitro culture and amplification: 1) can be mixed with epithelial stem cells to reconstruct hair; 2) the injection can be injected into hair for resting period to promote hair growth.
The cell sorting system can easily realize commercialization, and makes substantial contribution to the anti-alopecia industry in China.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a prior art expression of Prom1/CD133 in hair follicles;
epi, epidermis; ORS, out root sheath, outer root sheath; mx, matrix, wool matrix
DF, dermal fibroplast, dermal fibroblasts; DP, total dermal papillas cells, total dermal papilla cells; TAC, transit amplification cells, transiently proliferating cells; neg, mix of unlabeled cells; HF-SC, hair follicule muscle cell, hair follicle stem cells; HF-ORS, ORS minus stem cell, ectotheca stem cell; ZZ-DP, zig-zag DP, zigzag dermal papilla of hair follicles; G/AA-DP, Guard/awl and auchene DP, protects hair follicle/conical follicle dermal papilla cells; G-DP, Guard DP, protection of hair follicle dermal papilla cells; AA/ZZ-DP, awl and auchene/zigzag DP, cone follicle/zigzag dermal papilla cells of hair follicles;
FIG. 2 is a flow chart of a control group of example 1 of the present invention;
FIG. 3 is a flow chart of DP cell sorting of mouse dorsal skin hair follicles with LEPR antibodies according to example 1 of the present invention;
FIG. 4 is a diagram of the reconstructed back hair of a nude mouse according to example 2 of the present invention;
FIG. 5 shows an LEPR in example 2 of the present invention+DP cells can significantly induce hair patterns.
FIG. 6 is a fluorescence image of the LEPR antibody of the present invention at mouse and rat specific marker DP.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
"LEPR" as referred to herein refers to the leptin receptor encoding gene,the protein encoded by the gene can recognize and transport leptin. "LEPR+DP "refers to the sorted FITC positive cell population. "LEPR-DP "refers to the sorted FITC negative cell population.
Example 1
The invention provides a method for sorting dermal papilla cells based on a rabbit monoclonal antibody, which comprises the following specific steps:
step 1: preparation of LEPR Rabbit monoclonal antibody
1.1 immunization and serum detection
Immunization: immunizing two New Zealand white rabbits with LEPR recombinant protein antigen; immune site: subcutaneous multiple injections.
And (3) serum detection: the ELISA method detects the serum titer. The qualified standard of the immunity titer is OD1:64000>0.8。
And performing WB \ FC \ ICC \ IHC detection on the ELISA qualified serum.
1.2 in vitro culture of B cells and supernatant screening
Taking rabbit spleen, separating B cell, plating, culturing in vitro, adding cell growth factor and feeder cell, culturing for 10-15 days, and detecting supernatant.
Taking culture supernatant, carrying out primary screening, and selecting positive clones aiming at specific antigens.
The supernatant of ELISA positive clone was verified by WB \ FC \ ICC \ IHC.
All positive clones were selected, cell lysates prepared and stored at-80 ℃.
1.3 cloning recombination
Taking Top10 colibacillus clone for antibody recombination. The variable region sequences of the heavy and light chains were adjusted using specific PCR primers.
After sequencing verification, plasmid construction is performed, and small upgraded particles are prepared for transfection testing.
After transfection and positive detection of supernatant ELISA, WB \ FC \ ICC \ IHC verification is carried out.
And determining the positive clone antibody recombinant plasmid pair.
1.4 antibody production
And (4) preparing the large quality-improved particles of the optimal clone.
And (5) carrying out transfection production.
The transfection supernatant was subjected to ProA affinity column purification of antibody and buffer exchange, i.e., substitution of Gly-HCl with PBS, tris buffer, and final storage of PBS.
Step 2: separate DP cells of skin hair follicles of mice born for 30 days
Reagent: precooled 1 XPBS buffer, room temperature 1 XPBS buffer, calcium-free pancreatin stock, Collagenase (Collagenase), DMEM with 4% serum, 4% PBS-F (fetal bovine serum added to 1 XPBS at a concentration of 4%). PI (propidium iodide, concentration 1.5mg/ml)
Consumable material: a 40 μm cell filter screen, a 50mL centrifuge tube, a 1.5mL EP tube, a 10cm petri dish, a 6cm petri dish, sterile forceps, sterile scissors, and an electric shaving razor.
The technical scheme adopted for rapidly sorting the DP cells of the hair follicles of the mammals on a large scale comprises the following steps:
specifically labeling hair follicle dermal papilla cell (DP cell) suspension with the LEPR rabbit monoclonal antibody prepared in the step 1, specifically binding the primary antibody with the fluorescent secondary antibody, and finally sorting and collecting the DP cells labeled with the fluorescent secondary antibody in a large scale by using a flow cytometer, so that a large amount of DP cells can be collected in the shortest time.
The method comprises the following steps:
step a, preparing a suspension of hair follicle DP cells, and preparing a single cell suspension by adopting an enzyme digestion method, such as pancreatin and collagenase, specifically: the whole operation environment is operated in a sterile environment
Mice were sacrificed by cervical dislocation. (the selected mice have their hair in the growth phase, and typically B6 mice born for 30 days, i.e., C57BL/6 mice, available from Shanghai Jersey laboratory animals Co., Ltd.)
The skin area was scratched and the hair was shaved.
The skin is washed.
A10 cm petri dish was prepared in advance, and a pre-cooled 1 XPBS buffer was placed on ice. The skin was cut out and placed in a petri dish and washed clean with 1 × PBS buffer.
The dermis was facing up and the fat layer on the dermis side was torn off with forceps. Care was taken not to destroy the follicle.
3mL of 0.25% collagenase were placed in a 6cm petri dish and preheated to 37 ℃. The skin was digested in collagenase at 37 ℃. Digestion time (in a shaker at 37 ℃ C., 25rpm) was 0.5 h-2 h. The well digested skin is transparent.
A new 6cm petri dish was prepared and placed in 5mL of room temperature 1 XPBS buffer. The skin was transferred to a new petri dish and the collagenase was gently washed off. Repeat 2 times.
The PBS buffer was aspirated off, 3mL of 0.25% pancreatin was added, and the mixture was digested at 37 ℃ for about 30min at 25rpm on a shaker.
After digestion, transfer to ice. The latter operations were all on ice or in a 4 ℃ environment.
The skin was minced (cut into pieces of approximately 2mm by 2 mm).
The digestion was stopped by adding 3mL of DMEM and pipetting 10-12 times with a 10mL pipette.
The cell suspension was passed through a 40 μm cell filter and the filtrate was transferred to a 50mL centrifuge tube. (operation on Ice)
The petri dish was washed with DMEM, the wash passed through a 40 μm cell filter, and the filtrate was transferred to a 50mL centrifuge tube.
Centrifuge at 1500rpm, 4 ℃ for 5 min. The supernatant was aspirated off.
The cells were resuspended in precooled PBS-F buffer and counted. The suspension was gently blown down with 2ml PBS-F buffer solution and then resuspended with 3ml PBS-F buffer solution. (the number of cells extracted from the skin of the back of adult mice is approximately 2-5X 107cells/mL)
Step b FACS sample preparation
The cell suspension from step a was divided into multiple tubes (1.5 mLEP tubes) with the cell concentration of the experimental group at 2X 107cells/mL, control cell number 106The volume was adjusted to 800. mu.L. In the control group, the primary antibody was not added, and only the secondary antibody and PI (propidium iodide, which allows staining of DNA from dead cells, so that PI dyes can be used to distinguish dead and live cells). The remaining was used for cell sorting. (control and experimental groups were treated simultaneously in the following steps.)
Step c specific binding of LEPR monoclonal antibody to DP cells
All cell samples were centrifuged at 250g/1500rpm, 4 ℃ and 5 min.
Aspirate 500. mu.L of cell suspension into a 1.5mL EP tube, add primary antibody and incubate at 4 ℃ for 1h, and spin in a spinner. LEPR 1: 100 dilution. (dilution ratio considering the number of cells and solution volume) 500. mu.L of PBS-F buffer was added to a 1.5mLEP tube, and the cells were washed upside down.
250g/1500rpm, 4 ℃, 5min centrifugation, suction supernatant.
Add 500. mu.L PBS-F buffer for resuspension.
Add fluorescent secondary antibody and incubate for 30min, 4 ℃. Dark environment, spin in the spinner. (the information of the secondary antibody is Alexa 488-conjugated affinity double Anti-Rabbit, Jackson immune Research, the concentration is 1.5mg/ml after the configuration according to the instruction, the working concentration is 1: 200 dilution)
Add 500 u L PBS-F buffer to 1mLEP tube, upside down washing cells.
250g/1500rpm, 4 ℃, 5min centrifugation, suction supernatant.
The cells were resuspended by adding PBS-F buffer and placed on ice.
Adding PI about 5min before sorting, and mixing. PI 1: 1000 dilution.
Sterile EP tubes were prepared prior to sorting, and PBS-F was prepared for use as a wash flow channel.
Step d DP cell sorting
And (4) sorting DP cells at a high speed by sorting conditions of FITC positive and PI negative gating.
FIG. 2 is a control flow chart of the present invention. Under conditions of no primary antibody, plus secondary antibody, almost no DP cells were sorted. FIG. 3 is a flow chart of DP cell flow for sorting mice back skin follicles with LEPR antibodies of the present invention. The efficiency of sorting was 1.74% and the cell yield of one adult mouse was approximately 2-5X 107Therefore, the amount of DP cells obtained from one adult mouse sorting was about 348000-870000 cells.
Example 2 verification of LEPR+Activity of DP cells
Sorting LEPR+DP cells were cultured for 3 passages, cell amount 1425000, and neonatal mouse epidermal cells, cell amount 4000000, mixed and transplanted on the back of nude mice, as shown in FIG. 4A, with long hairs and arrows pointing to the regenerated hairs. FIG. 4B shows only the addition of neonatal murine epidermal cells, 4000000 cells, no hair growth. FIG. 4C sort LEPR-DP cells were cultured for 3 passages, with cell mass of 1850000, and neonatal mouse epidermal cells, with cell mass of 4000000, mixed and transplanted onto the backs of nude mice without hair growth.
As shown in FIG. 5, C57/B6 mice of 40 days of age (telogen) were shaved of back hair, LEPR+DP cells were cultured for 3 passages and 1X 106Cells were injected into the right side of the back of the mouse, left side was not injected as a control, and LEPR was additionally set-Negative cells and PBS injection control. It was observed that DP cells could significantly induce hair growth.
Example 3 cell sorting system based on specific recognition of LEPR monoclonal antibody has broad spectrum, and can be used for other mammals, not only for separating mouse dermal papilla cells, but also for separating other mammals with scientific and economic value, such as rat and human dermal papilla cells.
As shown in fig. 6A, fluorescence images were made of the binding of antibodies to LEPR with antibodies coupled with fluorescein FITC, after the antibodies specifically labeled mouse hair follicle dermal papilla cells. FIG. 6B is a fluorescence image of the specific labeling of rat hair follicle dermal papilla cells by the LEPR antibody, followed by binding of the antibody conjugated with fluorescein FITC to the LEPR antibody. The scale bar is 20 microns.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (9)
1. Use of a cell sorting system based on specific recognition of LEPR monoclonal antibodies, wherein the cell sorting system comprises the following components: the kit comprises a LEPR monoclonal antibody, a fluorescent secondary antibody and hair follicle dermal papilla cells, wherein the LEPR monoclonal antibody is used for specifically marking the hair follicle dermal papilla cells, and the fluorescent secondary antibody is used for specifically marking the LEPR monoclonal antibody.
2. The use of claim 1, wherein the cell sorting system is a method of sorting dermal papilla hair follicles comprising the steps of:
(1) preparing the hair follicle dermal papilla cell suspension by adopting an enzyme digestion method,
(2) specifically labeling hair follicle dermal papilla cells in the cell suspension with an LEPR monoclonal antibody,
(3) the fluorescent secondary antibody is specifically combined with the LEPR monoclonal antibody,
(4) and (4) sorting hair follicle dermal papilla cells according to the sorting condition set by cross-gating the emission wavelength of the fluorescent secondary antibody.
3. Use according to claim 2, wherein step (1) is embodied as the following step:
(1.1) selecting mice with hair in the growth period, killing the mice by a neck-breaking method,
(1.2) scratching the skin area, shaving off the hair, washing the skin,
(1.3) preparing a 10cm culture dish in advance, putting precooled 1 XPBS buffer solution on ice, cutting skin, putting the skin in the culture dish, washing the 1 XPBS buffer solution,
(1.4) the dermis was faced up, the fat layer on the dermis side was torn off with forceps without damaging the hair follicle,
(1.5) placing 3mL of 0.25% collagenase in a 6cm culture dish, preheating to 37 ℃, placing the skin in collagenase for digestion at 37 ℃, wherein the digestion time is 0.5-2 h, the fully digested skin is transparent,
(1.6) preparing a new 6cm dish, placing 5mL room temperature 1 XPBS in advance, transferring the skin to the new dish, washing off collagenase gently, repeating 2 times,
(1.7) sucking off PBS, adding 3mL of 0.25% pancreatin, placing in a shaking table at 37 ℃, digesting for 20-30min at 25rpm, transferring to ice after digestion, and then operating on ice or in an environment at 4 ℃,
(1.8) cutting the skin into small pieces of 2mm multiplied by 2mm,
(1.9) adding 3mL of DMEM containing 4% serum, terminating the trypsinization, pipetting 10-12 times in total volume with a 10mL pipette to form a cell suspension,
(1.10) passing the cell suspension through a 40 μm cell filter, transferring the filtrate to a 50mL centrifuge tube,
(1.11) washing the culture dish with DMEM, passing the washing solution through a 40 μm cell filter, transferring the filtrate to a 50mL centrifuge tube,
(1.12) centrifuging at 4 ℃ for 5min at 1500rpm, sucking off the supernatant,
(1.13) resuspend the cells in pre-cooled PBS-F buffer and count, gently blow well with 2mL PBS-F buffer, then resuspend with 3mL, suspension of dermal papilla cells of hair follicle.
4. The use according to claim 1, wherein the LEPR monoclonal antibody is prepared by a process comprising the steps of:
(i) two New Zealand white rabbits are immunized by LEPR recombinant protein antigen, the serum titer is detected by an ELISA method,
(ii) b cell in vitro culture and supernatant screening positive clones aiming at specific antigen,
(iii) a recombinant LEPR monoclonal antibody is disclosed,
(iv) and (3) preparing a large-quality-improved particle of the target clone, then carrying out transfection production, carrying out ProA affinity column purification on the transfection supernatant to obtain an LEPR monoclonal antibody, carrying out buffer solution replacement, namely replacing Gly-HCl with PBS and tris buffer solution, and finally storing the LEPR monoclonal antibody in the PBS buffer solution.
5. The use of claim 4, wherein in step (i) the site of immunization is subcutaneous multi-site injection and the eligibility criterion for the immune titer is OD1:64000>0.8。
6. The use according to claim 4, wherein step (ii) is in particular: taking rabbit spleen, separating B cells, plating, culturing in vitro, adding cell growth factor and feeder cells, culturing for 10-15 days, and detecting supernatant; taking the supernatant, carrying out primary screening, and selecting positive clones aiming at specific antigens; performing WB \ FC \ ICC \ IHC verification on the supernatant of the ELISA positive clone; all positive clones were selected, cell lysates prepared and stored at-80 ℃.
7. The use according to claim 4, wherein step (iii) is specifically performed by antibody recombination using the clone Top 10; using specific PCR primers to call variable region sequences of heavy chains and light chains; after sequencing verification, plasmid construction is carried out, and small upgraded particles are prepared for transfection test; after transfection and positive detection of supernatant ELISA, WB \ FC \ ICC \ IHC verification is carried out; and determining the positive clone antibody recombinant plasmid pair.
8. A kit for sorting hair follicle dermal papilla cells, which comprises a LEPR monoclonal antibody, a fluorescent secondary antibody and a PBS buffer solution, wherein the LEPR monoclonal antibody specifically marks the hair follicle dermal papilla cells, and the fluorescent secondary antibody specifically marks the LEPR monoclonal antibody.
9. The application of hair follicle dermal papilla cells specifically identified based on LEPR monoclonal antibody in hair growth.
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