Peripheral blood MNCs were incubated with Fc blocking reagent (Miltenyi Biotec) and stained as previously described. 17 (link),19 ,22 (link) “Fluorescent minus one” (FMO) gating controls were also used to ensure proper gating of positive events.17 (link),19 Briefly, cells were incubated with titred antibodies for 30 minutes at 4°C, washed twice in PBS with 2% fetal bovine serum (FBS), fixed in 1% paraformaldehyde (Tousimis), and run within 72 hours of fixation on a BD LSRII flow cytometer (BD, Franklin Lakes, NJ, USA) equipped with a 405nm violet laser, 488nm blue laser and 633nm red laser. Data were acquired uncompensated and exported as FCS 3.0 files, and analyzed utilizing FlowJo software, version 9.7.2 (Tree Star, Inc).
Paraformaldehyde (pfa)
Manufactured by BD
Sourced in United Kingdom, United States
Paraformaldehyde is a solid, white, crystalline compound used as a fixative in histology and cell biology applications. It is a polymer of formaldehyde that can be used to preserve and stabilize biological samples for microscopic analysis.
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Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Pe anti human asc clone hasc 71, by BioLegend (2 mentions)
Fitc anti human nlrp3, by R&D Systems (2 mentions)
Streptavidin pe, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by BD (2 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (2 mentions)
Rabbit anti flag antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (2 mentions)
Black 96 well plate, by Corning (2 mentions)
Af546 goat anti hamster igg, by Thermo Fisher Scientific (2 mentions)
Triton x, by Thermo Fisher Scientific (2 mentions)
Ly6g fitc antibody clone 1a8, by BioLegend (2 mentions)
Alf 161 armenian hamster anti mouse il 1α, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse cd63 clone epr21151, by Abcam (2 mentions)
Af647 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Vectashield antifade mounting media with dapi, by Vector Laboratories (2 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Fc blocking reagent, by Miltenyi Biotec (1 mentions)
31 protocols using paraformaldehyde (pfa)
1
Multicolor Flow Cytometry Analysis of PBMCs
2
Measuring HIV Envelope Antibody Binding
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The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: 69T1RevEnv Cells from Dr. Joseph Dougherty32 (link) and CEM.NKR CCR5+ cells from Dr. Alexandra Trkola.50 (link), 51 (link), 52 (link) HIV envelope expression on HeLa cells was measured 7 days and 2 passages after removing from tetracycline. CEM.NKr.CCR5 cells were incubated with 1.5 μg of HIV-1 YU2 gp120 recombinant protein (MyBioSource.com , San Diego, CA, USA) per 1 million CEM cells for 75 min. HIV envelope-expressing HeLa cells or CEM.NKr.CCR5 cells coated with HIV-1 gp120 were fixed with 4.2% paraformaldehyde (BD Biosciences, San Jose, CA, USA) for 20 min at 4°C. Cells were washed in chilled fluorescence-activated cell sorting (FACS) buffer (PBS + 2% FBS) and incubated in supernatant containing secreted antibody from both nontransduced and transduced cell supernatant for 1 h at room temperature. Cells were washed an additional two times in FACS buffer and incubated with goat anti-human IgG (H+L) fluorescein isothiocyanate (FITC) (Life Technologies, Carlsbad, CA, USA) for 30 min at room temperature before analysis by flow cytometry.
3
BrdU Incorporation Quantification in Mice
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BrdU was solubilized in 10 mN NaOH / H2O. 4 mg BrdU (Sigma) in 200 μl PBS was injected intraperitoneally into each mouse 2.5-3 hours before sacrifice37 (link),41 ,42 (link). Control mice were injected with 200 μl of PBS alone. For quantitation of BrdU incorporation, after isolation and RBC lysis, cell suspensions were fixed in 4% paraformaldehyde (BD) at room temperature for 10 minutes, then washed in 5% FBS followed by PBS. Cells were permeabilized in 100 μl of 0.125% Dawn dishwashing detergent (Procter and Gamble) in 1x PBS for 15 minutes at room temperature, then washed twice with 1% FBS in PBS. Cells were resuspended in DNase buffer (10% FBS in 1 mM CaCl2 and 0.1 mM MgCl2) containing 150U of DNase per sample and incubated at 37°C for 45 minutes before washing twice in PBS. Samples were then resuspended in Fc blocking buffer and incubated with antibody cocktail for analysis by flow cytometry.
4
Multiparametric Flow Cytometry Immunophenotyping of PBMCs
5
Flow Cytometric Analysis of Breastmilk Cells
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Flow cytometric analysis was conducted in cells isolated from freshly expressed breastmilk collected from two breastfeeding women according to Hassiotou et al. 201225 (link). In short, isolated cells were incubated with fixative (1.5% Paraformaldehyde: BDH, Poole, England; 1% Sucrose: Merck, Darmstadt, Germany in phosphate buffered saline/PBS: Invitrogen, Mulgrave, Victoria) for 10 minutes followed by washing in PBS and were then centrifuged for 30 seconds. Permeabilisation occurred by adding Tween 20 (USB, Cleveland, USA) 0.05% for 10 minutes to the cells. Primary antibodies (Table S2 ) were added to the cells in Tween 20 0.05% with 2% fetal bovine serum (Borogen, Essendon, Australia) and incubated for 1 hour in the dark at 4 °C following washing. Cells were washed twice and incubated with 1:200 secondary antibody solution (Table S2 ) as above for 30 minutes in the dark at 4 °C. Cells were washed twice in PBS/Tween 0.05%, and fixative was added. Appropriate negative internal controls were also prepared (no primary antibody). Data acquisition was done with a FACS Calibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com ) and data analysis was done with FlowJo.
6
Measuring Spike-Specific T Cell Responses
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We measured spike-specific CD4+ and CD8+ T cell responses by activation-induced marker (AIM) expression as previously described (Sabatino et al., 2022 ). Briefly, PBMCs were thawed, washed, and rested for 2 h at 37 °C prior to start of assay. PBMCs were stimulated for 24 h with 1 µg/ml spike peptide pools (JPT Peptide Technologies) or vehicle control (0.2% DMSO). Folllowing stimulation, cells were washed and stained with the following antibody panel: CD4 Alexa 488 (OKT4), CD8 Alexa 700 (SK1), OX-40 PE-Dazzle 594, (ACT35), CD69 PE (FN-50), CD137 BV421 (4B4–1), CD14 PerCP-Cy5.5 (HCD14), CD16 PerCP-Cy5.5 (B73.1), CD19 PerCP-Cy5.5 (HIB19) (all BioLegend) and live/dead dye eFluor506 (Invitrogen). Cells were washed with FACS wash buffer, fixed with 2% paraformaldehyde (BD), and stored in FACS wash buffer in the dark at 4 °C until ready for flow cytometry analysis. The same gating strategy was employed as before (Sabatino et al., 2022 ).
AIM-positive T cells were defined by the co-expression of OX-40 and CD137 by CD4+ T cells and co-expression of CD69 and CD137 by CD8+ T cells. AIM expression following spike peptide pool stimulation was subtracted from AIM expression following stimulation with no peptide negative control to yield the frequencies of spike-specific T cells.
AIM-positive T cells were defined by the co-expression of OX-40 and CD137 by CD4+ T cells and co-expression of CD69 and CD137 by CD8+ T cells. AIM expression following spike peptide pool stimulation was subtracted from AIM expression following stimulation with no peptide negative control to yield the frequencies of spike-specific T cells.
7
Tissue Histology and Collagen Quantification
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Tissue samples were fixed in 4% paraformaldehyde (BD Biosciences), dehydrated, and embedded in paraffin. Tissue blocks were sectioned for staining with hematoxylin and eosin staining or Masson’s trichrome staining, examined under a BX51 microscope (Olympus, Tokyo, Japan), and imaged using a DP71 digital camera (Olympus). Total collagen content was reported as the percentage of the total tissue area positively stained with aniline blue, as determined using ImageJ (National Institutes of Health, Bethesda, MD, United States) software.
8
Immunohistochemical Characterization of Cardiac Cells
9
Sputum Induction and Cell Analysis
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Sputum induction was performed as previously described.30 (link) The supernatants were kept frozen at –70°C until further analysis. For immunocytochemistry, cytospins were fixed with 4% paraformaldehyde (BDH Ltd., Poole, UK) and stored at –20°C. Total cell counts were recorded with a hemocytometer, using Kimura stain. Cell viability was determined by Trypan blue exclusion before cytospins were undertaken. The slides were stained with May-Grunwald-Giemsa stain, and differential cell counts were made by a blinded observer. Four hundred inflammatory cells were counted on two slides for each sample in a blinded manner. Differential cell counts are expressed as the percentages of total inflammatory cells. Samples with cell viability of greater than 70% and less than 30% squamous cell contamination were considered adequate for analysis.
10
Flow Cytometry Analysis of Genetically Modified T Cells
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A FACSCanto (BD Biosciences, San Jose, CA, USA) instrument was used to acquire immunofluorescence data, which were analyzed with CellQuest (BD Biosciences). FlowJo v.7 (FlowJo, Ashland, OR, USA) was used for final data analysis and graphic representation. Isotype controls were immunoglobulin G1-fluorescein isothiocyanate (IgG1-FITC; BD), IgG1-phycoerythrin (IgG1-PE; BD), IgG1-peridinin chlorophyll protein (IgG1-PerCP; BD), and isotype Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Genetically modified T cells were detected using a CD19-PE antibody (BD) along with CD8-PerCP, CD4-Pacific Blue, CCR7-FITC, and CD45RA-AF750. Forward and side scatter gating were used to discriminate live cells from dead cells.
Cells were collected and washed once with PBS (Sigma, St. Louis, MO, USA) containing 1% FBS (GE Healthcare Life Sciences HyClone Laboratories; FACS buffer) prior to the addition of antibodies. Cells were incubated with antibodies for 30 min on ice in the dark, washed once, and fixed in FACS buffer containing 0.5% paraformaldehyde (BD Biosciences) prior to analysis.
Cells were collected and washed once with PBS (Sigma, St. Louis, MO, USA) containing 1% FBS (GE Healthcare Life Sciences HyClone Laboratories; FACS buffer) prior to the addition of antibodies. Cells were incubated with antibodies for 30 min on ice in the dark, washed once, and fixed in FACS buffer containing 0.5% paraformaldehyde (BD Biosciences) prior to analysis.
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