KDAC6 (a gift from Eric Verdin, Addgene plasmid #13823)[25 (link)], KDAC7 (aa521-942), KDAC8, and KDAC8HA were cloned into pFastbac1 (Life Technologies) From pJExpress vectors with the TEV protease cleavage site and His6 tag. Constructs were transformed into DH10Bac E. coli cells to produce bacmids containing KDAC8 [26 (link)]. Bacmids were purified and transfected into Sf9 cells using Cellfectin II (Life Technologies) as described elsewhere [27 (link)]. Baculovirus from these transfections was amplified in Sf9 cells, then used to infect High Five insect cells in suspension in Express Five SFM (Gibco). At 72 hours post-infection, cells were pelleted and frozen at −20 °C until lysis.
Express five sfm
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Express Five SFM is a compact, benchtop flow cytometer designed for rapid and precise cell analysis. It features a fixed-alignment optical system and up to five laser excitation sources, enabling the detection and quantification of a wide range of cellular parameters. The core function of the Express Five SFM is to provide researchers with a powerful and user-friendly tool for multiparameter flow cytometry analysis.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Glutamax, by Thermo Fisher Scientific (6 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Sf 900 3 sfm, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Penicillin streptomycin mixture, by Lonza (3 mentions)
L glutamine, by Lonza (3 mentions)
Sodium pyruvate, by Lonza (3 mentions)
Cellfectin 2, by Thermo Fisher Scientific (2 mentions)
Effectene transfection reagent, by Qiagen (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Cacl2, by Lonza (2 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement, by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
L glutamin, by Thermo Fisher Scientific (1 mentions)
29 protocols using express five sfm
1
Cloning and Expression of KDAC Enzymes
2
Cell Culture Protocols for Drosophila, Mouse ESCs, and Human Fibroblasts
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Drosophila S2 cells (Thermo Fisher R69007) were cultured at 25 °C in serum free medium (Express Five SFM, Gibco), supplemented with 20 mM l -Glutamin (GlutaMAX, Gibco). Cells were passaged with 10% of conditioned medium each passage. Cells were maintained at a density of 1–12million ml−1.
Mouse embryonic stem cells were cultured in a humidified incubator at 37 °C and 5% CO2. Cells were grown on Attachment Factor (Thermo Fisher) in 2i medium (KnockOutTM Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 15% KnockOutTM Serum Replacement (Gibco), 0.1 mM MEAA (Gibco, non-essential aminoacid), 1 nM Na-Pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 4 mM l-Glutamin (GlutaMAX, Gibco), 5 μg ml−1 Insulin (Sigma), 50 U ml−1 Pen-Strep, 200 U ml−1 LIF (ESGRO), 1 μM PD0325901 (StemGent), 3 μM CHIR99021 (StemGent).
Human fibroblast cell lines were established from skin biopsies of confirmed Koolen de-Vries syndrome patients and healthy controls. Cells were maintained in in a humidified incubator at 37 °C and 5% CO2 in DMEM (GlutaMAX supplement, Life Technologies), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 10% foetal calf serum (FCS). All fibroblast lines tested negative for Hepatitis B, Hepatitis C, HIV and human Herpes virus 4 and 8. Fibroblasts were passaged at ~90% confluency.
Mouse embryonic stem cells were cultured in a humidified incubator at 37 °C and 5% CO2. Cells were grown on Attachment Factor (Thermo Fisher) in 2i medium (KnockOutTM Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 15% KnockOutTM Serum Replacement (Gibco), 0.1 mM MEAA (Gibco, non-essential aminoacid), 1 nM Na-Pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 4 mM l-Glutamin (GlutaMAX, Gibco), 5 μg ml−1 Insulin (Sigma), 50 U ml−1 Pen-Strep, 200 U ml−1 LIF (ESGRO), 1 μM PD0325901 (StemGent), 3 μM CHIR99021 (StemGent).
Human fibroblast cell lines were established from skin biopsies of confirmed Koolen de-Vries syndrome patients and healthy controls. Cells were maintained in in a humidified incubator at 37 °C and 5% CO2 in DMEM (GlutaMAX supplement, Life Technologies), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 10% foetal calf serum (FCS). All fibroblast lines tested negative for Hepatitis B, Hepatitis C, HIV and human Herpes virus 4 and 8. Fibroblasts were passaged at ~90% confluency.
3
Drosophila Cell Culture Protocols
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Schneider’s Drosophila medium (Gibco, ThermoFisher Scientific), supplemented with 10% fetal bovine serum (Gibco, ThermoFisher Scientific) and 1% penicillin–streptomycin (Gibco, ThermoFisher Scientific) was used to culture Drosophila S2 cells. Express Five SFM (Gibco, ThermoFisher Scientific) supplemented with 20 mM GlutaMAX (Gibco, ThermoFisher Scientific) and 1% penicillin–streptomycin was used to grow D.Mel-2 cells.
4
Co-Immunoprecipitation of Protein Complexes in S2 Cells
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S2 cell culture and western blotting were carried out as described below. Myc-Prd1, Flag-α-Ada, Flag-Rab5, Khc-Flag, and Imac-HA expression vectors were generated by Gateway cloning. S2 cells were cultured at 25°C in Express Five SFM (Gibco) medium supplemented with 1% L-glutamine. For transfection, S2 cells were plated at a density of 2 × 106 cells per 35-mm dish 1 d before transfection. After 24 h culture, around 0.1–0.5 μg of each expression plasmid was transfected into the S2 cells using Effectene Transfection Reagent (Qiagen); cells were harvested 48 h post-transfection. Transfected S2 cells were homogenized with lysis buffer (25 mM Tris pH 8/27.5 mM NaCl/20 mM KCl/25 mM sucrose/10 mM EDTA/10 mM EGTA/1 mM DTT/10% [v/v] glycerol/0.5% Nonidet P40) with protease inhibitors (Complete, Boehringer; PMSF 10 mg/mL, sodium orthovanadate 10 mg/mL). The supernatants were used for IP with anti-Myc, anti-Flag, or anti-HA overnight at 4°C followed by incubation with protein A/G beads (Pierce Chemical Co.) for 2 h. Protein A/ G beads were washed four times using cold PBS. Bound proteins were separated by SDS-PAGE and analyzed by western blotting with anti-Myc, anti-Flag, and anti-HA HRP-conjugated antibody. Co-IP experiments were repeated three times.
5
Cell Culture Protocols for Various Cell Lines
6
Culturing Drosophila S2 Cells
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Drosophila S2 cells were cultured in Schneider’s Drosophila medium (Gibco, Thermo Fisher Scientific, 11720-034), supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, 10082147) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, 15140122) at 25°C. Drosophila S2 cells adjusted to serum-free growth medium were cultured in Express Five SFM (Gibco, Thermo Fisher Scientific, 10486025) supplemented with 20 mM GlutaMAX (Gibco, Thermo Fisher Scientific, 35050061) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, 15140122).
7
Baculovirus-Mediated Recombinant Protein Production
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Baculovirus generation and amplification were performed in Sf9 cells (CRL-1771, American Type Culture Collection), a clonal isolate of Spodoptera frugiperda Sf21 cells, and grown in Trichoplusia ni medium-formulation Hink insect cell medium (Gemini Bioproducts) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), penicillin (100 U/ml)–streptomycin (100 μg/ml) solution (Gibco), and 0.1% (v/v) Pluronic F-68 (Gibco). High Five cells (BTI-TN-5B1-4, B85502, Thermo Fisher Scientific) were grown in Express Five SFM (Gibco) supplemented with 16 mM l -glutamine (Gibco) and used for recombinant HA and NA production (58 (link)). Both adherent cell lines were grown at 27°C.
Adherent Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells were grown in modified Eagle’s medium (MEM) containing 10% (v/v) FBS and penicillin (100 U/ml)–streptomycin (100 μg/ml) solution in a humidified incubator at 37°C and 5% CO2.
Adherent Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells were grown in modified Eagle’s medium (MEM) containing 10% (v/v) FBS and penicillin (100 U/ml)–streptomycin (100 μg/ml) solution in a humidified incubator at 37°C and 5% CO2.
8
Cell Culture and Transfection Protocols
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Hela cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (PAA) and 2 mM l -glutamine (Gibco) at 37°C and 5% CO2. S2 cells stably expressing pMT-Gal4 were cultured in Express Five SFM (Gibco) containing 2 mM l -glutamine (Gibco) and puromycin (concentration 10 µg/ml; Invitrogen) at 25°C. 24 h before fixing, cells were induced with Copper(II) sulfate pentahydrate (Sigma-Aldrich) at the concentration of 700 µM. Transfection of plasmids was performed using Fugene HD (Roche) according to the manufacturer’s protocol. Hela cells were treated with either 500 nM cytochalasin D or latrunculin A for 15 min at 37°C. S2 cells were treated with 100 nm rapamycin for 20 min at 25°C.
9
Insect Cell Lines for Baculovirus and Protein Production
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Insect cells used for the baculovirus preparation were Spodoptera frugiperda 21 (Sf21) (source EMBL), grown in Sf-900™ III SFM media (Gibco, 12658027), as published previously (Roostalu et al., 2018 (link)). Insect cells used for recombinant protein expression were Trichoplusia ni High Five insect cells, grown in Express Five™ SFM (Gibco, 10486025).
10
Culturing and Transfecting Drosophila S2 Cells
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S2 cell lines were obtained from the Drosophila Genome Resource Center (Bloomington, IL). S2 cells were maintained in Express Five SFM (Gibco) supplemented with 1% penicillin/streptomycin and 9% L-glutamine. Cells were removed from the flask using a cell scraper and passaged to maintain a density of approximately 106–107 cells/mL. S2 cells were transferred to SF900 SFM (Gibco) prior to transfection with Cellfectin II (Invitrogen). Transfections were performed according to Cellfectin II protocol in a final volume of 3 mL in a T-25 flask containing 5×106 cells that were plated one hour before transfection. The total amount of DNA used in transfections was 2.5 ug.
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