BALF were briefly centrifuged at 150 x g and the supernatant was collected for analysis. Pelleted cells were resuspended in PBS and cells were counted using a hemocytometer prior to loading into an EZ Cytofunnel (Thermo Scientific) and centrifuged at 600 RPM for 10 minutes. Slides were fixed and stained using the Hema 3 kit (Thermo Scientific) and cell differentials were counted based on at least 200 cells per slide.
Ez cytofunnel
Manufactured by Thermo Fisher Scientific
The EZ cytofunnels are a laboratory equipment product designed for use in cell preparation and analysis workflows. They provide a simple and efficient method for collecting cell samples. The core function of the EZ cytofunnels is to facilitate the transfer and deposition of cells onto a slide or other surface for further processing and examination.
Automatically generated - may contain errors
Lab products found in correlation
Hema3 kit, by Thermo Fisher Scientific (3 mentions)
Duoset elisa kit, by R&D Systems (2 mentions)
Synergy ht plate, by Agilent Technologies (2 mentions)
Mouse mucin 5 subtype ac elisa kit, by MyBioSource (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Tyramide alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti n cadherin, by BD (2 mentions)
Tsa kit 24, by Thermo Fisher Scientific (2 mentions)
Vectastain abc hrp kit, by Vector Laboratories (2 mentions)
Anti cardiac troponin t ct3 clone, by Developmental Studies Hybridoma Bank (2 mentions)
Anti myosin heavy chain antibody mf20 clone, by Developmental Studies Hybridoma Bank (2 mentions)
Ncl l p53 cm5p, by Leica (2 mentions)
Shandon cytospin 4, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
7 protocols using ez cytofunnel
1
BALF Cell Differential Analysis
2
Analysis of BAL Cytokines and Mucins
3
Analysis of BAL Cytokines and Mucins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BAL fluids were harvested by 3 successive instillations of 500 μL of PBS into the lungs, and pooled BAL fluids were briefly centrifuged at 150 × g and supernatants was collected for analysis of cytokines and other secreted mediators. BAL levels of IL-33, IL-5, IL-13, IL-17A, IL-1β and amphiregulin (AREG) were assessed using DuoSet ELISA Kits (R&D Systems; DY3626, DY405, DY413, DY1399, DY401, DY989) according to manufacturer protocol and were read on a BioTek Synergy HT plate reader. MUC5AC in BAL was analyzed using a Mouse Mucin-5 Subtype AC ELISA kit (MyBioSource; MBS265057), according to the manufacturer’s instructions. Pelleted BAL cells were resuspended in PBS and counted using a hemocytometer prior to loading into an EZ Cytofunnel (Thermo Scientific) and centrifuged at 600 RPM for 10 minutes. Cytospin slides were fixed and stained using the Hema 3 kit (Thermo Scientific) and cell differentials were counted based on at least 200 cells per slide.
4
Immunostaining of Embryoid Bodies
5
Cytospin Immunocytochemistry of mCSCs and mBMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured mCSCs and mBMSCs were suspended at 5 × 105 cells/mL in PBS and 200 μL was loaded into EZ Cytofunnels (ThermoFisher, Carlsbad, CA) within a Shandon Cytospin 4 (ThermoFisher, Carlsbad, CA). Cells were spun down for 3 min on low acceleration and then fixed onto polylysine-coated slides using Shandon Cell-Fixx fixative (ThermoFisher, Carlsbad, CA). Cells were dried overnight and prepared for immunocytochemistry with phalloidin and DAPI at 1:10,000 for 5 min. Confocal imaging utilized a 63× oil objective (Leica, Buffalo Grove, IL), with zoom factor of 0.75, 1.1 Airy unit pinhole, and HyD emission bandwidth of 415–510 at power intensity of 1.5 W and gain of 150 V. The pixel dimensions were 0.241 μm in the x and y plane and 0.357 μm in the z-plane during z-stack acquisition; step size was optimum at 0.35 μm with 28–30 slices per z-stack.
6
Immunostaining of Embryoid Bodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Day 9 EBs were harvested and fixed with 4% paraformaldehyde at 4°C overnight. EBs were sent to ULAM (Unit for laboratory animal medicine) at University of Michigan for paraffin processing, embedding, and sectioning. Antigen unmasking on slides was using citric acid methods as prescribed previously (Gage and Camper, 1997 (link)). Immunostaining was performed by combining of VectaStain ABC-HRP kit (Vector laboratories) and TSA Kit #24, with HRP-Streptavidin and Alexa Fluor 568 Tyramide (ThermoFisher Scientific). The primary antibodies were used for staining: anti-N-cadherin (mouse/monoclonal/1:2000, BD), anti- cardiac troponin T (CT3 clone, mouse/monoclonal/1:100, Developmental Studies Hybridoma Bank) and anti-myosin heavy chain antibody (MF20 clone, mouse/monoclonal/1:100, Developmental Studies Hybridoma Bank). For cytospin, Day 2 WT and Wdr5KO EBs were harvested and trypsinized to single cell suspension. Cells were attached to EZ cytofunnels (ThermoFisher) slides using Cytospin 3 (Shandon). Cells were fixed with 100% methanol and immunostaining was performed using p53 antibody (NCL-L-P53-CM5P, Leica biosystems) at 1:100 dilution. Counter nuclear staining was performed with DAPI (Molecular Probes). Control sections were incubated without primary antibodies. 4 representative pictures from different field were recorded under fluorescence microscope (Olympus DP73).
7
Immunofluorescence Staining of Cell Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence was performed on cytospins (EZ Cytofunnels) (Thermo Scientific) for SC and on coverslips for adherent cell cultures. Samples were fixed with ice-cold methanol for 10 minutes at -20°C, blocked with a 3% BSA/PBS solution followed by incubation overnight at 4°C with the primary antibody. Secondary antibody was applied for 1 hour at room temperature; samples were mounted with DAPI-containing Fluoromount G (Southern Biotech) and analyzed on a confocal microscope (Zeiss LSM 510 Meta). To certify that the staining was positive throughout the whole spheroid, z-stacks were performed. The antibodies used are listed in S1A Table .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!