The captured cells were fixed and treated inside the device. 4% paraformaldehyde (PFA) diluted in PBS at 10 μl/min for 15 min was injected from inlet 1 to outlet 2, followed by PBS rinsing at 50 μl/min for 6 min. Similarly, 0.2% Triton X-100 in PBS was injected into the device at 10 μl/min for 10 min for permeabilization and 300 μl 1% BSA in PBS was introduced at 10 μl/min for 30 min to block out non-specific bindings. Afterwards, cells were stained with dye solutions (passing from inlet 1 to outlet 1). First, 600 μl immunostaining solution, containing 5 μg/ml Anti-Pan-cytokeratin (AE1/AE3) Alexa Fluor 488- (1:100 diluted in PBS; eBioscience SAS) and Anti-Human CD45 PE (1:20 diluted in PBS, eBioscience SAS, France), was injected at 10 μl/min for 1 h to stain tumor cells and WBCs, respectively. Then, the nucleus were stained by 4′,6-diamidino-2-phenylindole (DAPI,100 nM, Life Technologies) at 10 μl/min for 10 min. Finally, the device was rinsed by 300 µl PBS for 6 min and fluorescence images were obtained with an inverted optical microscope (Zeiss, Axiovert 200) equipped with a digital CCD camera (Evolution QEI).
Anti human cd45 pe
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
The Anti-Human CD45 PE is a lab equipment product used to identify and quantify CD45-positive cells in biological samples. It is a fluorochrome-conjugated monoclonal antibody that specifically binds to the CD45 antigen expressed on the surface of various hematopoietic cells.
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Lab products found in correlation
2 protocols using anti human cd45 pe
1
Immunostaining and Imaging of Captured Cells
2
Single-Cell CTC Analysis in NSCLC
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Blood samples from stage IV NSCLC patients and healthy donors were obtained with informed consent in accordance with the approved procedures under the institutional review board (IRB) guidelines. The project was approved by the Singhealth Centralised Institutional Review Board (CIRB).
Analysis of the primary tumor was performed as part of the routine procedure during patient recruitment. Sample workflow for single cell capture is illustrated insupplementary Fig. 5 with total processing time of 7.5 hours. Blood samples of 7.5 ml were extracted from each patient if possible. Primary CTC enrichment was carried out via the ClearCell FX system (Clearbridge Biomedics, Singapore) at the Singapore General Hospital. The mixed cell population was then stained with Anti-Human CD45 PE (eBioscience, Inc., CA, USA) and further depleted of WBCs using our device and recovered as single cells in PCR tubes. The additional primer pair for L858R is as follows.
Forward: 5′- CTAACGTTCGCCAGCCATAAGTCC-3′
Reverse: 5′- GCTGCGAGCTCACCCAGAATGTCTGG-3′
Amplicons were sent for direct sequencing after purification. Results were single blinded and compiled after analyzing the direct sequencing results of all isolated single cells.
Analysis of the primary tumor was performed as part of the routine procedure during patient recruitment. Sample workflow for single cell capture is illustrated in
Forward: 5′- CTAACGTTCGCCAGCCATAAGTCC-3′
Reverse: 5′- GCTGCGAGCTCACCCAGAATGTCTGG-3′
Amplicons were sent for direct sequencing after purification. Results were single blinded and compiled after analyzing the direct sequencing results of all isolated single cells.
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