clemente-associates goat anti-mouse IgG说明书
1、制备带有结合抗HLA-G的纳米颗粒。在操作前一天，用小鼠单克隆抗HLA-G抗体（BD）将磁性纳米颗粒与山羊抗小鼠IgG（Clemente Assoc.）偶联。结合100μL无菌PBS、10μL抗体（0.5 mg/mL）和10μL纳米粒。在4°C的冷藏室摇杆上搅拌过夜。第二天，通过首先添加900μL无菌PBS，然后磁化粒子10分钟，去除所有液体，去除未结合抗体。
2、初始宫颈内样本到达ThinPrep溶液。400 x g的颗粒细胞持续5分钟。将细胞颗粒重新放入12-13 mL无菌PBS中，最终体积为14 mL。
将样品穿过插入15 mL离心管的250μm组织过滤器，以去除大块粘液和细胞团。
3、通过离心和在14 mL无菌PBS中重新悬浮细胞，将宫颈样品清洗2次。
4.最后一次清洗细胞后，将样品重新悬浮在1.4 mL无菌PBS中。向样品中添加100μL抗HLA-G涂层磁性纳米颗粒（制备于#1）以分离滋养层细胞。在4°C的冷藏室摇杆上培养过夜。
5、隔夜孵育后，将滋养层细胞在4℃的磁铁（DynaMag Spin magnet；Life Technologies）上分离5分钟。使用磁铁，在4℃下用1 mL无菌PBS清洗滋养层细胞3次（在移液前让纳米粒子磁化10分钟）。最终移除未结合的细胞后，将捕获的细胞在4℃下重新悬浮在100μL无菌PBS中。
6、取一小份分离的细胞悬液，计数分离的胎儿细胞，计算回收的胎儿细胞总数。使用血细胞仪对第一次清洗的母细胞进行计数。
为了检查细胞的纯度，用抗-hCG。
确定荧光灯数量hCG阳性细胞和总细胞（DAPI标记）。
派生%hCG阳性。
| 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.