The naïve TauP301S-Venus HEK293 cell line was treated with preformed 1N4R Tau fibrils as published previously (23 (link)). Briefly, cells were plated in a 6-well plate in Opti-MEM reduced serum medium and GlutaMAX supplement (Gibco). The next day, the cells were treated with 100 nm preformed Tau fibrils and 10 μl Lipofectamine 2000 (Invitrogen) diluted in Opti-MEM. After 1 h of incubation time, equal volumes of complete DMEM were added. 3 days later, the cells were washed and resuspended in cell sorting buffer (1× PBS plus 0.8 mm EDTA, 0.5% [v/v] FCS) before carefully passing the cells through a 35-μm cell strainer (Corning). Focus-containing cells were sorted based on the intracellular distribution of Venus fluorescence (concentrated intensity signal versus diffuse distribution) on a FACS Aria IIIu (Becton Dickinson) with a 530/30-nm filter at the ZMBH FACS facility. Using these stringent sorting conditions, 7500 cells (2.5% of the original cell population), which contained a highly concentrated rather than a diffuse intracellular Venus signal, were collected in complete DMEM, expanded, and frozen. The presence of TauP301S-Venus foci before and after freezing/thawing was confirmed by fluorescence microscopy.
35 μm cell strainer
Manufactured by Corning
Sourced in United States
The 35-μm cell strainer is a laboratory tool used to filter and separate cell suspensions. It is designed to allow the passage of single cells while retaining larger cellular aggregates, debris, and clumps. The strainer has a pore size of 35 micrometers, making it suitable for a variety of cell types and applications.
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Lab products found in correlation
Rnase inhibitor, by Takara Bio (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Glass dounce tissue grinder, by Merck Group (3 mentions)
Tryple express, by Thermo Fisher Scientific (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Cell counter r1, by Olympus (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Tc20 automated cell counter, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Cd4 clone rm 4 5, by Thermo Fisher Scientific (2 mentions)
100 μm cell strainer, by Corning (2 mentions)
Red blood cell lysing buffer hybri max, by Merck Group (2 mentions)
Clone tonbo 2, by Thermo Fisher Scientific (2 mentions)
H 2kb clone af6 88, by Thermo Fisher Scientific (2 mentions)
H 2db clone 28 14 8, by Thermo Fisher Scientific (2 mentions)
Anti mouse isotype igg2a clone ebm2a, by Thermo Fisher Scientific (2 mentions)
Cd8α clone 53 6, by Thermo Fisher Scientific (2 mentions)
22 protocols using 35 μm cell strainer
1
Generation of TauP301S-Venus HEK293 Cell Line
2
Cell Ploidy Analysis by Flow Cytometry
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The ploidy of cells was analyzed by flow cytometry. Cultured cells were detached from a dish using PBS containing 0.025% Trypsin and 0.01% EDTA. Cells were fixed in 70% ethanol at −20 °C and then washed with PBS containing 2% FBS. The cell suspension was centrifuged at 200× g for 5 min twice. The pellet was suspended in PBS containing 2% FBS and propidium iodide (PI; Dojindo Laboratory) and then filtered through a 35-μm cell strainer (Corning Inc.). Cells were analyzed on a FACS Canto II Flow Cytometer (BD Biosciences). At least 4 × 104 events were recorded for each analysis. Data were analyzed using Kaluza Flow Cytometry Software version 1.1 (Beckman Coulter, Inc., Brea, CA).
3
Mammosphere Formation Assay Protocol
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Cells in monolayer culture were disassociated with TrypLE (Gibco) and passed through a 35-μm cell strainer (Corning), resulting in single-cell suspensions. Cells were then stained with trypan blue and counted using a Cell Counter R1 (Olympus) to determine the concentration of viable cells. We plated 500 viable cells per well in an ultra-low attachment 96-well plate in 100 μL of the mammosphere media (MEGM media with 1% methylcellulose containing 10 ng/mL EGF, 20 ng/mL FGF, and 4 μg/mL heparin) as described.34 (link) Mammosphere media was replenished every 3 days. Cells were either pretreated in monolayer cultures or treated during the mammosphere assay as indicated. After 12 days of treatment, each well was imaged using a Cytation3 Plate Reader and Imager (BioTek) at 4x magnification and stitched together into a tile using the BioTek Gen5 program. Spheres larger than 70 μm in diameter were considered mammospheres and counted manually.34 (link)
4
Isolating G0-G1 Phase 4T1 Cells
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4T1 cells with 2N DNA content (G0–G1) were isolated following the treatment of DMSO or 1 μM FiVe1 for 72 h. Cells were stained with 5 μg/mL Hoechst 33342 in monolayer culture for 2 h in a 37°C cell culture incubator. Cells were then dissociated with TrypLE (Gibco) and passed through a 35-μm cell strainer (Corning). The concentration of viable cells was determined by staining with trypan blue and counting on a Cell Counter R1 (Olympus). Cells were sorted using a MoFlo Astrios Cell Sorter (Beckman Coulter) at the MD Anderson Flow Cytometry & Cellular Imaging Facility. The DMSO-treated cells, with a normal distribution of cells in the cell cycle, were used to gate for the 2N population. The cells in the 2N population were isolated for subsequent experiments.
5
Nuclei Isolation for Single-Cell Analysis
6
Nuclei Isolation from Tissue Samples
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Nucleus isolation was done as previously described8 (link). Briefly, tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 ml of ice-cold nuclei EZ lysis buffer. The sample was then incubated on ice for 5 min, with an additional 3 ml of cold EZ lysis buffer. Nuclei were centrifuged at 500g for 5 min at 4 °C, washed with 5 ml ice-cold EZ lysis buffer and incubated on ice for 5 min. After centrifugation, the nucleus pellet was washed with 5 ml nuclei suspension buffer (NSB; consisting of 1× PBS, 0.01% BSA and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). Isolated nuclei were resuspended in 2 ml NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per µl was used for loading on a 10x channel.
7
Cellular Uptake of Labeled PRXs
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4T1 cells were seeded in 24-well plates at a density of 5 × 104 cells/well and incubated overnight. The cells were then cultured in a medium containing Cy5.5-labeled PRXs for 3 h at 37 °C. Subsequently, the cells were harvested and collected via centrifugation (1500 rpm, 3 min, 4 °C). The cells were then washed with D-PBS containing 0.1% BSA and passed through a 35 μm cell strainer (Corning, Corning, NY, USA). The fluorescence intensity of the cells was measured using a BD FACS Canto II flow cytometer as described above.
8
Thawing and Processing Cryopreserved PBMCs
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Cryopreserved human peripheral blood mononuclear cells (PBMCs) and isolated peripheral blood CD4+, CD8+, CD14+, CD19+ and CD56+ cells were purchased from AllCells (see Table S1 for catalog numbers and donor information). Cells were quickly thawed in a 37°C water bath, rinsed with culture medium (Iscove’s Modified Dulbecco’s Medium (IMDM) (ATCC) supplemented with 10% FBS and 1% Pen/Strep) and then treated with 0.2 U/μL DNase I (Thermo Fisher Scientific) in 10mL of culture medium at 37°C for 30 min. After DNase I treatment, cells were washed with medium once and then twice with ice-cold 1x PBS (Gibco) + 0.1% BSA (MilliporeSigma). Cells were then filtered with a 35 μm cell strainer (Corning) and cell viability and concentration were measured with trypan blue on the TC20 Automated Cell Counter (Bio-Rad). Cell viability was greater than 80% for all samples.
9
Cryopreserved PBMC and Cell Isolation
10
Isolation of Intact Nuclei from Mouse Hearts
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Mice hearts were harvested and frozen after sacrificing them. Frozen tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 mL of ice-cold nuclei EZ lysis buffer. The sample was then incubated in ice for 5 min with 3 mL of cold EZ lysis buffer. Nuclei were centrifuged at 500 g for 5 min at 4°C, washed with 5 mL ice-cold EZ lysis buffer, and incubated on ice for 5 min. After centrifugation, the nuclear pellet was washed with 5 mL nuclei suspension buffer [NSB; consisting of 1% PBS, 0.01% BSA, and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)]. Isolated nuclei were resuspended in 2 mL NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per μl was used for loading onto a 10x channel.
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