Cells harboring high copy mRFP1 expression plasmid, BBa_J04450-pSB1C3, were grown aerobically in M9 glucose medium for 12 h at 37 °C, with 0.5 mM of isopropyl β-d -1-thiogalactopyranoside induction. Then, 1 ml of the cell culture was diluted in 9 ml of PBS and cells were dissociated using a round bottom polystyrene test tube with cell strainer snap cap (Corning). Then, samples were analyzed on an S3e Cell Sorter (Bio-Rad). A total of 100,000 events were collected and analyzed by the FlowJo software (FlowJo). Contour plots showing gating strategy are provided in Supplementary Figure 19 .
Cell strainer snap cap
Manufactured by Corning
The Corning Cell Strainer Snap Cap is a laboratory filtration device designed to separate cells or other particulate matter from a solution. It features a snap-on cap that securely attaches to a variety of laboratory containers, allowing for easy handling and processing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Falcon round bottom polystyrene test tubes, by Corning (2 mentions)
S3e cell sorter, by Bio-Rad (1 mentions)
Cd69 h1.2f3, by BioLegend (1 mentions)
Zombie red, by BioLegend (1 mentions)
Cd86 gl 1, by BioLegend (1 mentions)
Falcon tube, by Corning (1 mentions)
Attune nxt software, by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Facsuite, by BD (1 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Lsr 2 cytometer, by BD (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Liberase, by Roche (1 mentions)
Fixable viability dye, by BioLegend (1 mentions)
Egm 2 medium, by Lonza (1 mentions)
Falcon test tube, by Corning (1 mentions)
Lipofectamine ltx plus, by Thermo Fisher Scientific (1 mentions)
Stempro accutase, by Thermo Fisher Scientific (1 mentions)
11 protocols using cell strainer snap cap
1
Flow Cytometry Analysis of mRFP1 Expression
2
Analyzing Lymph Node Immune Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 24, 48, and 72 h p.i., virus-draining popliteal LNs and non-draining brachial LNs were harvested from recipient mice and homogenized using a Corning™ Falcon™ tube with Cell Strainer Snap Cap (5 ml). 2–5 × 106 cells/well were plated on 96-well V-bottom plates and Fc receptors were blocked with 2.4G2 mAb for 20–30 min at 4°C. Cells were stained with fluorochrome—conjugated mAbs CD8 (53–6.7), CD4 (GK1.5), and CD69 (H1.2F3) from BioLegend (San Diego, CA) in 50–100 μl FACS buffer for 20 min at 4°C. Alternatively, cells were stained with biotinylated mAb against CD25 (PC61) followed by secondary staining with fluorochrome-conjugated streptavidin for 20 min at 4°C. For the proliferation assay at 72 and 120 h p.i., cells were stained for with fluorochrome-conjugated mAbs against CD8 (53–6.7) and CD4 (GK1.5). For DC activation analysis at 24 h p.i., cells were stained with fluorochrome—conjugated against mAbs CD11c (N418), MHC class II (M5/114.15.2), CD80 (16–10A1), and CD86 (GL-1) from BioLegend (San Diego, CA) in 30 μl FACS buffer for 30 min at 4°C. Zombie Red™ (BioLegend) was used as viability dye. After staining, the fixation was performed with 1% PFA for 20 min at 4°C. Samples were analyzed using AttuneTM NxT Flow Cytometer and AttuneTM NxT Software (ThermoFisher), FlowJoTM 10 software (Treestar) and GraphPad Prism.
3
PD-L1 Expression Quantification by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with fluorescence-activated cell sorting (FACS) buffer (filtered 0.1% BSA in PBS) and stained with phycoerythrin (PE)-conjugated monoclonal antibody specific for PD-L1 (eBioscience, MIH1) or IgG (Miltenyi Biotec, 130-092-212). Cells were filtered using a Falcon 5 ml round bottom tube with a cell strainer snap cap (Corning, 352235). Flow cytometric analysis was performed with FACSVerse and FACSuite (BD Biosciences).
4
Lung Cell Isolation and FACS Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lungs were perfused through the right ventricle with 5 ml of PBS to remove blood. The lung tissue was enzymatically digested using 0.2 mg/ml Liberase TM (Roche) and 100U/ml Deoxyribonuclease I (Sigma, DN25) for 45 mins, and then passed through nylon mesh (70 µm pores) to obtain a single cell suspension as described42 (link),49 (link),50 (link). Red blood cells were lysed in ACK lysis buffer. To remove cell debris, single cell suspensions from the lung tissue were passed through cell strainer snap cap (Corning life sciences). Single cell suspensions were stained with fixable viability dye (Biolegend) followed by the incubation with TruStain FcX (Biolegend). To identify cell surface antigens, cells were stained with a mixture of fluorochrome-conjugated antibodies (Supplementary Table 7 ). Data were acquired using BD LSR II cytometer and FACSDiva 8.0 software. Compensation and data analyses were performed using FlowJo software (TreeStar version 10.5.1). The Foxf1-GFP reporter line containing GFP knocked into the endogenous Foxf1 gene locus was used to purify FOXF1+ gCAPs. Stained cells were separated using the five-laser FACS Aria II cell sorter (BD Biosciences). Sorted cells were first collected into EGM-2 medium (Lonza), and then washed once with the medium after centrifugation at 300 g for 5 mins.
5
Isolation of Murine Conjunctival and Lymph Node Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized by extensive cardiac perfusion. Two conjunctivae of one mouse were harvested by excising the eyelid and bulbar conjunctiva, combined, minced, and exposed to 0.9 ml of 1 mM CaCl2, 2 mg/ml collagenase D, 0.16 mg/ml DNaseI and 0.05 mg/ml Dispase II in HBSS for 45 min at 37°C with shaking. Cervical and submandibular lymph nodes were isolated, minced and exposed to 1mg/ml collagenase D for 30 min at 37°C with shaking. After collagenase treatment, conjunctiva and draining lymph node cells were separately filtered through a 40 μm filter using a 1 cc syringe plunger. Then conjunctival cells were filtered a final time using a Corning Falcon Test Tube with Cell Strainer Snap Cap (Corning 352235).
6
Efficient CRISPR-Cas9 Cell Subcloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For transfection-based editing, cells were seeded 24 h prior transfection. Transfections were performed at 60 to 80% confluency using Lipofectamine LTX PLUS (ThermoFisher Scientific, cat #15338100). Plasmid DNA with Cas9 endonuclease and the relevant sgRNA cloned into the GFP-containing backbone plasmid (Addgene, cat #48138) were used for expression. Then, 48 h post transfection, cells were washed with DPBS pH 7.4 (Gibco™, cat #14190094) and dissociated with StemPro™ Accutase™ (Gibco™, cat #A1110501) at 37 °C for 5 min. The resulting pellet was resuspended in fresh media and passed through a cell strainer snap cap (Corning, cat #352235). Positively transfected cells (GFP-positive) were subcloned into 96-well plates (Corning, cat #3595). Cell sorting was performed using purity mode at a flow rate of 100 on a fluorescence-activated cell sorter (FACS) Melody™ instrument (Beckmann-Dickinson) with stringent gating for the top 60% of the positive parent population (SI Appendix, Fig. S21 ). Electroporated cells were subcloned into 96-well plates using the same sorting parameters on a FACS Aria II™ instrument (Beckmann-Dickinson).
7
PBMC Isolation for Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5 ml Falcon polystyrene round-bottom tubes (Corning Life Sciences, cat. no. 352054).
5 ml Falcon polystyrene round-bottom tube, with cell strainer snap cap (Corning Life Sciences, cat. no. 352235).
PBS as staining buffer.
Peripheral blood mononuclear cells (PBMCs) from a young healthy male adult was isolated from a leukocyte reduction chamber (LRC) purchased from the Oklahoma Blood Institute, with Lymphoprep (StemCell Technologies). Oklahoma Blood Institute performed relevant informed consents for blood product use for research purposes. Because the chambers are a byproduct of platelet donation and do not cause additional risk to the donor, the ECU Institutional Review Board does not require board review of protocols. Protocols were approved by East Carolina University Institutional Biosafety Committee protocol #01–19. All the methods were carried out in accordance with relevant guidelines and regulations.
8
Flow Cytometry Analysis of Fluorescent Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected from 10 cm dishes and diluted in 1 mL PBS inside Falcon test tubes with cell strainer snap cap (Corning). 50,000 -100,000 events were acquired using a BD FACSAria III cytometer and data were processed using FlowJo (FlowJo, LLC). Events were gated by forward and side scatter in parallel to exclusion of the doublet cells, and median fluorescence values were then calculated. The FITC channel (488-530/30) was used to measure mTFP1 fluorescence, and APC channel (633-660/20) was used to measure mCarmine or mNeptune fluorescence.
9
Flow Cytometry Analysis of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 48 hr after transfection, cells were harvested, washed with phosphate-buffered saline (PBS), and strained into single-cell suspensions using Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Caps (Corning). Fluorescence (FITC and PE/Texas Red) was measured using a BD LSRFortessa Cell Analyzer (Roy J. Carver Biotechnology Center Flow Cytometry Facility, Univ. Illinois). A total of 20,000 events were recorded for each replicate, and data were analyzed using FlowJo v10 (FlowJo, LLC).
10
Flp-In T-Rex 293 Cell Cycle Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flp-In T-Rex 293 cells were washed twice in phosphate-buffered saline (PBS) and trypsinized. They were recovered in PBS and centrifuged at 300 × g for 5 min. Cell pellets were then resuspended in 500 µL of PBS and dropped in 1 mL of cold ethanol, while vortexing it. Cells were fixed at 4 °C for at least 30 min. After a centrifugation at 300 × g and a wash of PBS, cell pellets were resuspended in 500 µL of FxCycle PI/RNase Staining Solution (ThermoFisher Scientific) and further incubated at 37 °C for 30 min. Cells were finally filtered with Cell Strainer Snap Caps (Corning) and processed for analysis on an Accuri C6 flow cytometer (BD). Data were analyzed using FlowJo.
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!