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clemente-associates goat anti-mouse IgG說明書
1、制備帶有結(jié)合抗HLA-G的納米顆粒。在操作前一天，用小鼠單克隆抗HLA-G抗體（BD）將磁性納米顆粒與山羊抗小鼠IgG（Clemente Assoc.）偶聯(lián)。結(jié)合100μL無菌PBS、10μL抗體（0.5 mg/mL）和10μL納米粒。在4°C的冷藏室搖桿上攪拌過夜。第二天，通過首先添加900μL無菌PBS，然后磁化粒子10分鐘，去除所有液體，去除未結(jié)合抗體。
2、初始宮頸內(nèi)樣本到達(dá)ThinPrep溶液。400 x g的顆粒細(xì)胞持續(xù)5分鐘。將細(xì)胞顆粒重新放入12-13 mL無菌PBS中，最終體積為14 mL。
將樣品穿過插入15 mL離心管的250μm組織過濾器，以去除大塊粘液和細(xì)胞團(tuán)。
3、通過離心和在14 mL無菌PBS中重新懸浮細(xì)胞，將宮頸樣品清洗2次。
4.最后一次清洗細(xì)胞后，將樣品重新懸浮在1.4 mL無菌PBS中。向樣品中添加100μL抗HLA-G涂層磁性納米顆粒（制備于#1）以分離滋養(yǎng)層細(xì)胞。在4°C的冷藏室搖桿上培養(yǎng)過夜。
5、隔夜孵育后，將滋養(yǎng)層細(xì)胞在4℃的磁鐵（DynaMag Spin magnet；Life Technologies）上分離5分鐘。使用磁鐵，在4℃下用1 mL無菌PBS清洗滋養(yǎng)層細(xì)胞3次（在移液前讓納米粒子磁化10分鐘）。最終移除未結(jié)合的細(xì)胞后，將捕獲的細(xì)胞在4℃下重新懸浮在100μL無菌PBS中。
6、取一小份分離的細(xì)胞懸液，計(jì)數(shù)分離的胎兒細(xì)胞，計(jì)算回收的胎兒細(xì)胞總數(shù)。使用血細(xì)胞儀對(duì)第一次清洗的母細(xì)胞進(jìn)行計(jì)數(shù)。
為了檢查細(xì)胞的純度，用抗-?hCG。
確定熒光燈數(shù)量?hCG陽性細(xì)胞和總細(xì)胞（DAPI標(biāo)記）。
派生%?hCG陽性。
| goat anti Mouse IgG [H + L chains] | gam250hl | 250 nm 2 ml | gam50hl | 50 nm 2 ml |
| Rat anti Mouse IgG 1 | ram250 1 | 250 nm 2 ml | ram50 1 | 50 nm 2 ml |
| Rat anti Mouse IgG 2a | ram250 2a | 250 nm 2 ml | ram50 2a | 50 nm 2 ml |
| Rat anti Mouse IgG 2b | ram2502b | 250 nm 2 ml | ram502b | 50 nm 2 ml |
| Rat anti Mouse IgM | ram250m | 250 nm 2 ml | ram50m | 50 nm 2 ml |
TRIC Cell Protocol
1. Prepare nanoparticles with bound anti-HLA-G. One day before the procedure, incubate
magnetic nanoparticles coupled to goat anti-mouse IgG (Clemente and Assoc.) with
mouse monoclonal anti-HLA-G antibody (BD). Combine 100 μL sterile PBS, 10 μL
antibody (0.5 mg/mL), and 10 μL nanoparticles. Incubate overnight with mixing on the
rocker in cold room at 4°C. The next day, remove unbound antibody by first adding 900
μL sterile PBS and then magnetizing the particles 10 min before removing all liquid.
2. The initial endocervical sample arrives in ThinPrep solution. Pellet cells at 400 x g for 5
minutes. Resuspend the cell pellet in 12-13 mL sterile PBS to a final volume of 14 mL.
Pass the sample through a 250 μm tissue strainer inserted into a 15 mL centrifuge tube
to remove large pieces of mucus and cell clumps.
3. Wash the cervical sample 2 times by centrifugation and resuspension of cells in 14 mL
sterile PBS.
4. After last wash of cells
,
resuspend sample in 1.4 mL of sterile PBS. Add the entire 100 μL
of anti-HLA-G-coated magnetic nanoparticles (prepared in #1) to the sample to isolate
trophoblast cells. Incubate overnight on the rocker in cold room at 4°C.
5. After the overnight incubation, separate the trophoblast cells on the magnet (DynaMag-
Spin magnet; Life Technologies) for 5 minutes at 4oC. Wash the trophoblast cells 3 times
with 1 mL sterile PBS at 4oC
,
using the magnet (allow nano particles to magnetize 10
minutes for each wash before pipetting). After the final removal of unbound cells,
resuspend the captured cells in 100 μL of sterile PBS at 4oC.
6. Remove a small aliquot of the isolated cell suspension and count the fetal cells isolated
to calculate the total number of fetal cells recovered. Count the maternal cells from the
first wash
,
using the haemocytometer.
7. To check the purity of the cells, immunofluorescently label cells with anti-?hCG.
Determine number of fluorescent ?hCG positive cells and total cells (DAPI labeled).
Derive %?hCG positive.
