Wild type Jurkat cells (TIB-152), K562 cells (CCL-243), HEK293T cells (CRL-3216), and T2 (CRL-1992) cells were obtained from the ATCC. J76 cells, a TCR negative Jurkat cell clone, are a generous gift from Mark Davis Lab from Stanford. Jurkat, J76, K562 cells were maintained in RPMI 1640 media (Cytiva, SH30096.FS) supplemented with 10% FBS (Sigma, F0926) and 10uM Glutamax (Gibco, 35050061). T2 cells were maintained in DEME media (Cytiva, SH30243.FS) supplemented with 10% FBS and 10uM Glutamax. To established HLA-A*03:01 monoalleic antigen presenting cell line, WT K562 were electroporated with a sleeping beauty transposase plasmid (VectorBuilder pRP[Exp]-CMV>T7/SB100X) and a sleeping beauty transposon plasmid (VectorBuilder sleeping beauty backbone, VB230803–1359aaa) co-expressing HLA-A*03:01 gene, B2M gene, and mNeonGreen gene as described previously63 (link). The plasmids were constructed with VectorBuilder service (vector ID: VB900088–2243bzq). The electroporation was performed with Lonza 4D unit (Protocol CL-120) and 2ug total plasmid DNA per 1e6 cells. HLA-A*03:01 positive cells were sorted with Sony cell sorter SH800S based on the surface HLA level and GFP level (WT K562 cells have none to low HLA levels).
Sh30243
Manufactured by Cytiva
Sourced in United States
SH30243.01 is a laboratory centrifuge product manufactured by Cytiva. This centrifuge is designed to separate different components within a liquid sample based on their density and size. The product specifications and technical details are not available for inclusion in an unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (2 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 media, by Cytiva (1 mentions)
Glutamax, by Cytiva (1 mentions)
Streptomycin, by Corning (1 mentions)
Penicillin, by Corning (1 mentions)
L glutamine, by GE Healthcare (1 mentions)
Rko cell line, by American Type Culture Collection (1 mentions)
Hepes buffered saline, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cms8861, by HiMedia (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
8 protocols using sh30243
1
Generation of HLA-A*03:01 Expressing K562 Cells
2
BV2 Cell Culture Protocol
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BV2 mouse microglial cells (RRID: CVCL_0182) were grown in Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L glucose (Cytiva #SH30243.02) supplemented with 10% fetal bovine serum (GE Life Sciences #SH30910.03), 2 mM L-Glutamine (GE Life Sciences #SH30852.01), 100 units/mL Penicillin and 100 g/mL Streptomycin (Corning #30-001-CI) in a standard tissue culture incubator with 5% carbon dioxide, unless otherwise specified.
3
Primary Osteoprogenitor Isolation from Facet Joints
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Facet joints were cut into ≤1 cm bone chips with scissors. The bone chips were washed in serum-free Dulbecco’s modified Eagle medium (DMEM) (SH30243.01; Hyclone Laboratories, Logan, UT, USA) containing 1% penicillin–streptomycin (15140122; Gibco Laboratories, Gaithersburg, MD, USA) antibiotics and incubated in DMEM growth medium containing penicillin–streptomycin and 10% fetal bovine serum (15140122; Gibco Laboratories) at 37°C for 2 weeks to isolate primary OPs. Cell suspensions were filtered through a nylon mesh, washed in serum-free DMEM several times, and seeded for culture. All isolated OPs were assessed with mycoplasma negative using a polymerase chain reaction (PCR)-based method (Takara, 6601).
4
Osteogenic Differentiation of Primary OPs
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Osteogenic differentiation and its quantitative methods were previously reported [16 (link)20 (link, link, link, link)]. Briefly, primary OPs from the facet joints were seeded in DMEM growth medium (SH30243.01; Hyclone Laboratories) and then differentiated with osteogenic medium (ascorbic acid, β-glycerophosphate, and dexamethasone) into mature osteoblasts for indicated days. Matrix maturation of differentiation was assessed by alkaline phosphatase (ALP) staining and activity. Matrix mineralization were assessed by alizarin red (ARS), von Kossa (VON), and hydroxyapatite (HA) staining. Differentiation medium was changed every 3 days.
5
RKO Cell Line Culture Protocol
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RKO cell line was purchased from American Type Culture Collection (ATCC, USA) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; SH30243.01, Hyclone Laboratories Inc) with 10% fetal bovine serum (FBS; 16000-044, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (15140122, Gibco).
6
CGGBP1 Knockdown in HEK293T Cells
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HEK293T cells were transduced with a control (CT) or CGGBP1-targeting (KD) shRNA as described elsewhere [5 (link)]. These cells were maintained in DMEM (SH30243.01, Cytiva HyClone) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (10270106, Invitrogen) and selected with 0.3 μg/ml of the final concentration of puromycin (CMS8861; HiMedia). For the equality of transfection of the Control DNA, the cloned-in carrier DNA in the same vector was cotransfected in CT and KD using the calcium-phosphate transfection method. 1.25 μg of each of the Control DNA and the carrier DNA (cloned in pGEM-T Easy vector) and 20 μl of 2M CaCl2 were mixed. A final volume of 200 μl is attained with sterile autoclaved water. This mix was added dropwise in 200 μl of 2X HEPES buffered saline (51558; Sigma) with gentle agitation on a vortex mixer to form calcium phosphate-DNA precipitate. The precipitate was incubated at room temperature for 20 minutes and added drop-by-drop on approximately 2 × 106 cells per 10 cm dish. On the next day, the cells were processed for AbC G4-ChIP.
7
Schwann Cell Culture Protocols
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The mouse neuronal Schwann cell line, SW10 (CRL-2766), and the human Schwann cell line, hTERT ipn02.3 2λ (hTERT02.3, CRL-3392), were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). SW10 cells were then maintained at subconfluence in Dulbecco Modified Eagle’s Medium (DMEM, SH30243.01, Cytiva, Marlborough, MA, USA) supplemented with 1% (v/v) penicillin/streptomycin (30-022-CI, Corning, NY, USA) and 9% (v/v) fetal bovine serum (FBS, SH30919.03, Cytiva, Marlborough, MA, USA) in an incubator at 33 °C under a humidified atmosphere of 5% CO2. hTERT02.3 cells were incubated in DMEM added with 1% (v/v) penicillin/streptomycin, 9% (v/v) FBS, and 2 mM L-glutamine (25030-081, Gibco, Waltham, MA, USA) at 37 °C.
8
Chick Satellite Cell Differentiation
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The chick satellite cells are replaced with a differentiation medium that consists of 2 % HS and 1 % P/S in Dulbecco’s Modified Eagle Medium (DMEM, SH30243.01, Cytiva, USA) if they reach sufficient density (>70 %). To replace the medium, remove the culture medium and wash with 1X DPBS. After washing, they will be replaced with a differentiation medium, and muscle cells will be cultured for 72 h at 37 °C in a 5 % CO2 incubator. Differentiation media was incubated at a dose of 150 μL/cm2 with rotation every 3 days.
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