Barcode-drug mixtures for the valve module were prepared by diluting barcoded RT primers in FreeStyle media (Thermo Fisher) to 1 µM. Drugs dissolved in DMSO were added to their corresponding barcode at 2x the final concentrations (see supplementary materials). The barcode-drug mixtures were supplemented with either Cascade-Blue (Thermo Fisher) or Alexa-488 (Thermo Fisher) at 10 µM for monitoring purposes and subsequently aspirated into 5 ml luer-lock syringes (BD) connected with PTFE tubings using 27 G ¾ needles (BD). Barcode-drug mixtures for the autosampler-based injection were prepared in round bottom 96-well plates by diluting barcoded PCR primers to 4 µM in FreeStyle media and the corresponding drugs to 4x the final concentrations. Mixtures were supplemented with Alexa-488 at 10 µM and plates were sealed with adhesive qPCR seals (Thermo Fisher).
Freestyle media
Manufactured by Thermo Fisher Scientific
Sourced in United States
Freestyle media is a cell culture media product designed for the growth and maintenance of a variety of cell lines. It provides a defined, serum-free formulation that supports the proliferation of cells in suspension culture. The core function of Freestyle media is to facilitate the in vitro cultivation of cells in a controlled and standardized environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Hitrap, by Cytiva (4 mentions)
Hek293f, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
25 kd branched pei, by Polysciences (3 mentions)
Luer lock syringe, by BD (2 mentions)
Histrap hp column, by Cytiva (2 mentions)
Superose 6 column, by Cytiva (2 mentions)
Amicon ultra 100 kda cutoff, by Merck Group (2 mentions)
Hitrap ninta column, by GE Healthcare (2 mentions)
Amicon 10 000 kda mwco filtration unit, by Merck Group (2 mentions)
Superdex 200 16 600 column, by GE Healthcare (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Expi293f, by Thermo Fisher Scientific (2 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (2 mentions)
T25 or t75 flasks, by Thermo Fisher Scientific (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Pei max, by Polysciences (2 mentions)
Hek293 freestyle cells, by Thermo Fisher Scientific (2 mentions)
Hek293s gnti cells, by American Type Culture Collection (2 mentions)
42 protocols using freestyle media
1
Barcode-drug Mixture Preparation
2
Purification of SARS-CoV-2 S Protein Ectodomain
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The codon optimized SARS-CoV-2 2P S protein ectodomain construct (GenBank: YP_009724390.1 ) was C-terminally tagged with 8xHis and a twin Strep tag and cloned into the mammalian expression vector pcDNA 3.1 (Synbio). HEK293F cells were grown in suspension culture using FreeStyle media (ThermoFisher) at 37°C in a humidified CO2 incubator (8% CO2). Cells were transiently transfected at a density of 1 × 106 cells/ml using branched polyethylenimine (PEI) (Sigma) (Portolano et al., 2014 (link)). Media was exchanged after 24 h and supplemented with 2.2 mM valproic acid. Supernatant was harvested by centrifugation after 4 days, filtered and loaded onto a 5 mL HisTrap HP column (Cytiva). The column was washed with buffer (20 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole) and the protein was eluted with buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Purified protein was concentrated (Amicon Ultra 100 kDa cut off, Millipore Sigma) and loaded onto a Superose 6 column (Cytiva) equilibrated with GF buffer (20 mM Tris pH 8.0 and 150 mM NaCl). Peak fractions were pooled and concentrated to 1.3 mg/ml (Amicon Ultra 100 kDa cut off, Millipore Sigma).
3
Recombinant VHH Protein Production
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The B3-, and VHH control 1B7-IFNg coding sequences were sub-cloned into the mammalian expression vector pVRC and transiently transfected using polyethyleneimine into HEK293F cells cultured in FreeStyle media (ThermoFisher #12338018). Media containing secreted protein was harvested 6 days following transfection by centrifugation at 8000 g for 20 minutes at 4°C, then loaded onto a HiTrap NiNTA column (GE Healthcare) and washed with 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 200 16/600 column (GE Healthcare) in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80°C.
4
Comparative RNA Capture Strategies
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K562 cells were cultured as described in section “Preparation of cell suspensions”. On the day of the experiments, cells were washed in PBS and resuspended in FreeStyle Media (Thermo Fisher)”, supplemented with 1% FBS. For comparing Combi-seq and PolyA-based RNA capture strategies, 250 cells/μl were transferred into an Eppendorf tube and mixed with a 5 μl solution, containing the corresponding drugs or DMSO, together with Combi-Seq or PolyA-Seq barcodes suspended in FreeStyle Media, 1% FBS. After 16 h incubation at 37 °C and 5% CO2 atmosphere, cells were mixed with 15 μl of the solution containing lysis buffer, ligase, and reverse transcriptase as in the droplet experiment (see section “Picoinjection for cell lysis, barcode ligation and reverse transcription” for details).
5
Cell Culture Protocols for Various Cell Lines
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Suspension cells were grown in plain bottom, vented flasks (Thermo Fisher Scientific), adherent cells were grown in T25 or T75 flasks (Thermo Fisher Scientific). Cells were maintained at 37°C and 5% CO2. K562 (CCL-243; ATCC) cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS), 1% GlutaMax, and 1% penicillin/streptomycin. HEK293T (CRL-3216; ATCC) and LentiX cells were maintained in DMEM supplemented with 10% FBS, 1% GlutaMax, and 1% penicillin/streptomycin. K562 SunTag-VP64 (CRISPRa) cell line was a gift from M Bassik. NKL cells including NKL-KIR2DL1 and KIR3DL1 expressing NKL cells were a gift from P Parham. HEK293F (R79007; Thermo Fisher Scientific) were grown in FreeStyle media (12338018; Thermo Fisher Scientific). Expi293F (A14528; Thermo Fisher Scientific) cells were grown in Expi293 Expression Medium (Thermo Fisher Scientific). Insect Hi5 cells (Tni; Expression Systems, 94–002S) were grown in ESF 921 media (Expression Systems) with a final concentration of 10 mg l−1 of gentamicin sulfate (Thermo Fisher) at 27 °C and atmospheric CO2. Cell lines tested negative for mycoplasma (MycoAlert Mycoplasma Detection kit, Lonza).
6
Cell Line Maintenance Protocol
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Suspension cells were grown in plain-bottom,
vented flasks (Thermo); adherent cells were grown in T25 or T75 flasks
(ThermoFisher). Cells were maintained at 37 °C and 5% CO2. HEK293T (CRL-3216; ATCC), and LentiX cells were maintained
in DMEM supplemented with 10% FBS, 1% GlutaMax, and 1% penicillin/streptomycin.
Caco-2, YT1, A431, UT/7, BaF3, 3T3, and B16 cells were obtained from
ATCC and grown and maintained according to ATCC specifications. HEK293F
(R79007; ThermoFisher) were grown in FreeStyle media (12338018; ThermoFisher).
Expi293F (A14528; ThermoFisher) cells were grown in Expi293 Expression
Medium (ThermoFisher). Cell lines tested negative for mycoplasma (MycoAlert
Mycoplasma Detection kit, Lonza).
vented flasks (Thermo); adherent cells were grown in T25 or T75 flasks
(ThermoFisher). Cells were maintained at 37 °C and 5% CO2. HEK293T (CRL-3216; ATCC), and LentiX cells were maintained
in DMEM supplemented with 10% FBS, 1% GlutaMax, and 1% penicillin/streptomycin.
Caco-2, YT1, A431, UT/7, BaF3, 3T3, and B16 cells were obtained from
ATCC and grown and maintained according to ATCC specifications. HEK293F
(R79007; ThermoFisher) were grown in FreeStyle media (12338018; ThermoFisher).
Expi293F (A14528; ThermoFisher) cells were grown in Expi293 Expression
Medium (ThermoFisher). Cell lines tested negative for mycoplasma (MycoAlert
Mycoplasma Detection kit, Lonza).
7
Cell Culture Methodologies for Cell Lines
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HEK293F (R79007, Thermo Fisher) and HEK293S (CRL-3022) cells were grown at 37 °C, 70% humidity 8% CO2 at 130 rpm in Freestyle media (12338018, Thermo Fisher). Jurkat cells (TIB-152, ATCC) and K562 cells (CCL-234, ATCC) cells were grown at 37 °C in RPMI with 10 % FBS and penicillin/streptomycin (15140122, Gibco). The Lenti-X 293T cell line (632180, Takara Bio Inc.) was grown in DMEM (41966-029, Gibco) with 10 % FBS and penicillin/streptomycin.
8
Exosome Isolation from STX-S Cells
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STX-S cells were cultured in FreeStyle media (ThermoFisher, 12338018) in a Multitron incubator (Infors HT) at 37°C, 80% humidified atmosphere with 8% CO2 on an orbital shaker platform. Subsequently, cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using either Capricor’s lab scale or large-scale purification method. For lab scale: supernatant was subjected to concentrating filtration against a Centricon Plus-70 Centrifugal filter unit (Millipore, UFC710008), then subjected to size exclusion chromatography (SEC) using a qEV original SEC column (Izon, SP5). For large scale: supernatant was subjected to concentrating tangential flow filtration (TFF) on an AKTA Flux s instrument (Cytiva, United States) and then subjected to chromatography on an AKTA Avant 25 (Cytiva, United States).
9
Expression and Purification of VHH Proteins
10
Preparation of K562 Cell Suspension
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K562 cells (ATCC, CCL-243) were cultured in IMDM media (Thermo Fisher) supplemented with 10% FBS (Thermo Fisher) and 1% Penicillin-Streptomycin (Thermo Fisher). On the day of the experiments, cells were washed twice in PBS and resuspended in FreeStyle Media (Thermo Fisher) supplemented with 4% FBS. The concentration of the cell suspension was adjusted to 2 × 106 cells ml−1 and subsequently aspirated into a 3 ml luer-lock syringe (BD).
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