BMDCs of the indicated genotypes were surfaced labeled with an anti-I-Ab-APC antibody (BD Biosciences) for 30 min, washed in DC culture media (RPMI-10% FBS + GM-CSF + IL-4), and incubated for 30 min to allow internalization. Cells were then spun down and resuspended in FACS buffer stripping solution (PBS containing 2% BSA Fraction V [Sigma Aldrich] and 0.1% NaN3, pH 1.5) for 10 min on ice and washed in stripping solution. Cells were then washed in cold FACS buffer (pH 7.4 PBS containing 2% BSA Fraction V [Sigma Aldrich] and 0.1% NaN3) and resuspended in DC culture media. Resuspended DCs were then incubated for 0, 10, 20 and 40 min to allow resurfacing of the internalized I-Ab. Following incubation, cells again were spun down and resuspended in FACS buffer stripping solution for 10 min on ice and washed in stripping solution. Cells were then washed, resuspended in 500 µl FACS buffer and analyzed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed using FlowJo 8.8.7 software (Tree Star, Ashland, OR). The percentage of recycled I-Ab was measured using the equation (T0 – Tx)/T0×100. T0 represents the mean fluorescence of cells following the second acid strip at time zero and Tx is the mean fluorescence intensity of cells stripped at each time point. The acid stripping method was adapted from Sullivan et al. [16] (link), [23] (link).
Bsa fraction 5
Manufactured by Merck Group
Sourced in United States, Germany
BSA Fraction V is a highly purified bovine serum albumin (BSA) product commonly used in various laboratory applications. It serves as a protein standard, stabilizer, and blocking agent in various biochemical and cell culture procedures.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Nicotinamide, by Merck Group (5 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Exendin 4, by OriGene (3 mentions)
B27 supplement, by STEMCELL (3 mentions)
Pnpp one component substrate, by Southern Biotech (3 mentions)
Imject alum, by Thermo Fisher Scientific (3 mentions)
Tween 20, by Merck Group (3 mentions)
Hoechst 33258, by Merck Group (3 mentions)
Gentamicin, by Merck Group (3 mentions)
Hepes, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Hanks buffered saline solution, by Corning (2 mentions)
Accumax, by Merck Group (2 mentions)
Insulin transferrin selenium its, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Aprotinin, by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
75 protocols using bsa fraction 5
1
Quantifying I-Ab Receptor Recycling in BMDCs
2
Quantification of Infectious Human Rhinovirus
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Plaque assays quantitating infectious HRV were performed as previously described, with modifications (Lee et al., 2015b (link)). In brief, HRV-B14 virus stock or cell supernatants were serially diluted in DPBS containing calcium, magnesium, and 0.1% BSA fraction V (Sigma-Aldrich). 200 µl diluted samples were added in duplicate to confluent monolayers of H1-HeLa cells in 6-well plates. After virus adsorption for 1 h at room temperature, the cells were overlaid with 0.8% Noble agar (Sigma-Aldrich) in 1x P6 medium (1x sMEM [Gibco], 26.2 mM sodium bicarbonate, 40.6 mM magnesium chloride hexahydrate, and 0.1% BSA fraction V [Sigma-Aldrich]). Nutritive medium was then overlaid to obtain final concentrations of 1x P6 medium, 2 mM l -glutamine (Gibco), 1.2 mM pyruvic acid (Sigma-Aldrich), 2 mM oxaloacetic acid (Sigma-Aldrich), and 0.1% glucose (Corning). After incubation at 35°C for 2 to 3 d, monolayers were fixed with 10% buffered formalin (Sigma-Aldrich) for 15 min at room temperature, overlaid agar removed, and plaques visualized by staining with 0.1% crystal violet (Sigma-Aldrich) in 20% ethanol for 1 h. Plaques were counted and calculated as PFU/ml of original virus stock or cell supernatant.
3
Fatty Acid Uptake in Cell Lines
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ARPE19 cells and HEK293T cells were transduced overnight with mouse ELOVL4 adenovirus and treated with FA precursors as described in Logan et al. (9 (link)). Sodium salts of the FAs were conjugated with BSA fraction V (Sigma) in a ratio 2:1 (w/w) BSA:FA for 20:5n3 (EPA) and 1:1 (w/w) BSA:FA for 26:0, 28:0, 30:0, and 34:5n3. Cells were treated with 30 μg/ml of the FA in media for a period of 48 h (HEK293T) or 72 h (ARPE19) unless otherwise stated. Following treatment, cells were harvested, washed once in 0.1 M PBS containing 50 μM of BSA fraction V (Sigma) to sequester excess free FAs, and washed with PBS only. The cell pellets were stored at −80°C until further processed for lipid analysis.
4
Single-cell RNA-seq of chick hindbrain
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HH4 embryos electroporated with FoxD3-NC2:eGFP were cultured until HH12 ex ovo at 37°C, following which the hindbrain region spanning rhombomeres 6, 7, and 8 was dissected under a fluorescence microscope. For dissociation, several different conditions were tested (dissociation in a glass dish for 1 hr in Accumax (EMD Millipore), chemical dissociation on a nutator for 15, 30, and 45 min, and chemical dissociation with gentle pipetting for 15, 30, and 45 min). The quality of the single-cell suspension obtained was tested by a trypan blue-based live-dead staining. Accordingly, we pooled dissected tissue washed in chilled 1X DPBS and incubated it in Accumax cell dissociation solution for 15 min at 37°C with gentle mixing every 5 min. Dissociation was terminated using Hanks Buffered Saline Solution (HBSS) (Corning) supplemented with BSA Fraction V (Sigma; 0.2% w/v). The suspension was centrifuged at 300 g for 4 min to collect cells at the bottom, the supernatant was removed, and the pellet was resuspended in 1 mL HBSS-BSA. To remove cell debris and clumps, the 1 mL suspension was passed through a 20 µm filter in a clean hood. This suspension was loaded on a 10X Chromium chip A (v2) to generate GEMs. The library was prepared according to the manufacturer’s protocol and sequenced on the Illumina HiSeq platform using the paired end chemistry.
5
Redifferentiation of Human Islet Cells
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Human islets (Table 1 ) were received 2–6 days following isolation. Islets were dissociated into single cells. Cells were cultured as previously described [1 (link)] in CMRL 1066 medium containing 5.6 mM glucose and supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT); 100 units/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml gentamycin (PSG); 5 mg/ml amphotericin B; and 3.5 mg/ml L-glutamine. The cells were refed twice a week and split 1:2 once a week. 293T cells were cultured in DMEM supplemented with 10% FBS, PSG, and 3.5 mg/ml L-glutamine. Redifferentiation cocktail (RC), consisting of 1% BSA fraction V (Sigma), 1X insulin/transferrin/selenium (ITS, Invitrogen), D-Glucose (final concentration 25 mM), 8 nM exendin-4 (Acris), 8 nM activin A (Cytolab/PreproTech), 1X B27 supplement (Stem Cell Technologies), and 10 mM nicotinamide (Sigma), in CMRL 1066 medium supplemented with PSG, was prepared and applied to cells as previously described [27 (link)]. ALK5 inhibitor II (Enzo), and FOXO1 inhibitor AS1842856 (Millipore), were applied to cells every 48 hours at a final concentration of 0.1 μM.
6
Immunofluorescence Assay of Anti-AMSP-Fu35 Antibodies
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Anti-AMSP-Fu35 antibodies from mice and rabbits were tested for their abilities to recognize the native proteins within the parasite by immunofluorescence assays (IFA). Synchronized P. falciparum 3D7 schizont stage smears were air-dried and fixed with pre-chilled methanol at -20°C for 30 min. The fixed smears were blocked by 3% bovine serum albumin (BSA Fraction V; Sigma) in PBS pH 7.4 for 2hrs at room temperature and then washed twice for 5min each with PBS. Slides were co-immunostained with AMSP-Fu35 sera in combination with sera of individual components viz PfAARP, PfMSP-311 or PfMSP-119. All the sera were used at a dilution of 1:100 except PfMSP-311 sera which was used at a dilution of 1:50 in 1% BSA in PBS-T (diluent) for 1hr. After washing thrice with PBS, the slides were incubated further with anti-mouse and anti-rabbit IgG conjugated to Alexa 488 and Alexa 594 respectively at 1:300 in diluent for 1hr at room temperature. Slides were subsequently washed, air dried and mounted with DAPI antifade (Invitrogen). Fluorescence and co-localization was examined under a Nikon SE300 confocal microscope with 100 X oil immersion objective.
7
Epinephrine-Stimulated Lipolysis Measurement
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The measurement of basal and epinephrine-stimulated lipolysis was performed as described previously [39 (link)]. The distal parts of the epididymal adipose tissue were incubated in Krebs-Ringer phosphate buffer containing 3% bovine serum albumin (BSA) fraction V (Sigma, USA) at 37°C, pH 7.4, with or without epinephrine (1.37 μM). The tissue was incubated for 2 h and the NEFA concentrations were measured.
8
Immunogenicity of Recombinant gp120 from Different Cell Lines
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Each group of C57Bl/6 mice was immunized with gp120 prepared from either 293F or
CHO-S cells. The recombinant gp120 (50 µg) was mixed with Imject Alum (Thermo
Fisher Scientific) and injected subcutaneously. Mice were boosted with same dose of
antigen at the indicated days along with Alum. Serum gp120 specific Ig levels in
these mice were analyzed by ELISA. Briefly, 96 well ELISA plates (Nalgene, Nunc) were
coated with gp120 (0.8 μg/well) overnight at 4°C, washed and blocked
with 1% BSA fraction V (Sigma–Aldrich), serum titers were then added to the
plates and incubated 4 hr at 4°C. After washing alkaline phosphatase-labeled
goat anti-mouse IgM or IgG isotype specific antibodies were added for 2 hr at room
temperature (SouthernBiotech, Birmingham, AL). After washing, PNPP one component
substrate (SouthernBiotech) was used to detect the amount of antibody bound.
CHO-S cells. The recombinant gp120 (50 µg) was mixed with Imject Alum (Thermo
Fisher Scientific) and injected subcutaneously. Mice were boosted with same dose of
antigen at the indicated days along with Alum. Serum gp120 specific Ig levels in
these mice were analyzed by ELISA. Briefly, 96 well ELISA plates (Nalgene, Nunc) were
coated with gp120 (0.8 μg/well) overnight at 4°C, washed and blocked
with 1% BSA fraction V (Sigma–Aldrich), serum titers were then added to the
plates and incubated 4 hr at 4°C. After washing alkaline phosphatase-labeled
goat anti-mouse IgM or IgG isotype specific antibodies were added for 2 hr at room
temperature (SouthernBiotech, Birmingham, AL). After washing, PNPP one component
substrate (SouthernBiotech) was used to detect the amount of antibody bound.
9
Phosphatidylcholine Extraction and Analysis
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Purified phosphatidylcholine from soybean lecithin (Phospholipon 90G, CAS-number 97281-47-5) was purchased from Lipoid (Ludwigshafen, Germany). Trizma base, HEPES, Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), glycine, ammonium persulfate, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, 2-mercaptoethanol, Hoechst 33258, and BSA-fraction V were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PVDF membranes, high performance chemiluminescence film, and enhanced chemiluminescence- (ECL-) Plus are from Amersham Biosciences (GE Healthcare, Piscataway, NY, USA). Mini-Protean apparatus for SDS-polyacrylamide electrophoresis, miniature transfer apparatus, acrylamide, bis-acrylamide, and TEMED were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Anti-EGFR (1005) antibody and secondary antibodies conjugated with HRP were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA, USA). Antibodies anti-phospho-mTOR Ser2448, anti-mTOR, anti-p44/42 MAP kinase (ERK 1/2), and anti-phospho-p44/42 MAP kinase Thr202/Tyr204 were from Cell Signaling Technology Inc. (Beverly, MA, USA). Cy3-conjugated secondary antibody against rabbit polyclonal immunoglobulins was from Jackson ImmunoResearch Laboratories, Inc. Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific, Pierce Protein Research Products (Rockford, IL, USA).
10
Lung Tissue Dissociation Protocol
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The lungs were surgically removed, rinsed in ice-cold PBS and transferred into a gentleMACS C tube (Miltenyi Biotec, 130-096-334) containing tissue digestion buffer (TDB). TDB consisted of 1× penicillin/streptomycin (Thermo Fisher Scientific, 15140122), 2× antibiotic–antimycotic (Thermo Fisher Scientific, 15240062), 1 mM sodium pyruvate (Thermo Fisher Scientific, 1360070), 1× MEM non-essential amino acids solution (Thermo Fisher Scientific, 11140035), 0.13 Wunsch units (WU) Liberase (Merck, 5401127001) and 160 U DNase I (Sigma-Aldrich, D4527-10KU) made up in KnockOut DMEM (Thermo Fisher Scientific, 10829018). Each sample was further dissociated with the gentleMACS Octo Dissociator system with heaters using the 37C_m_LDK_1 protocol. The cell suspension was filtered through a 70-µm cell strainer, and the cell strainer was rinsed once with 10 ml of wash buffer (WB; containing 0.5% bovine serum albumin (BSA; BSA Fraction V, Sigma-Aldrich, 10735096001) and 2 mM EDTA (Thermo Fisher Scientific, 14190-094) in PBS). Cells were collected via centrifugation (4 °C, 300g, 5 min).
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