Lacrimal glands, regional draining cervical lymph nodes (CLN) were harvested from the experimental murine groups at the end of the study, and single cell suspensions were prepared. In order to increase total cell counts multiple tissue samples were combined. Cells were stained with the following fluorescent conjugated antibodies: anti-CD4 eFluor450 (RM4–5), anti-CD25 APC-Cy7 (PC61), anti-FoxP3 PE (FJK-16s) (eBioscience, San Diego, CA and BD Bioscience, San Jose, CA). For intracellular cytokine staining, the cells were placed in a 96-well plate overnight in cell culture media with Cell Stimulation Cocktail (plus protein transport inhibitor, eBioscience) and stained with anti-IFN-γ FITC (XMG 1.2) (eBioscience, San Diego, CA). Absolute counting beads were used for the lacrimal gland (ThermoFisher Scientific, Waltham, MA). Stained cells were analyzed using FlowJO (Ashland, OR).
Absolute counting beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Absolute counting beads are small, uniform particles used in flow cytometry to determine the absolute count of cells or other particles in a sample. They provide a known number of beads per unit volume, allowing the calculation of the absolute number of target cells or particles in the sample.
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Lab products found in correlation
Easysep mouse monocyte isolation kit, by STEMCELL (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Pen strep glut, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Benchmark Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Cell stimulation cocktail plus protein transport inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ fitc xmg 1.2, by Thermo Fisher Scientific (1 mentions)
Celltrace violet dye, by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
96 well u bottom plate, by BD (1 mentions)
Canto 2 flow cytometer, by FlowJo (1 mentions)
Stemspan sfem 2, by STEMCELL (1 mentions)
Recombinant mouse m csf, by BioLegend (1 mentions)
Transwell plate, by Corning (1 mentions)
C57bl 6 wt mice, by Charles River Laboratories (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
16 protocols using absolute counting beads
1
Immune Cell Analysis in Murine Lacrimal Glands
2
Quantifying Culturable and VBNC Cells
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The counts of culturable cell were determined using the standard plate count method according to Su et al. (2015b) (link). Each sample was serially diluted 10-fold with 0.9% (w/v) physiological saline (sodium chloride solution) and incubated on LB agar at 30°C for 48 h. The culturable cell counts of R. biphenylivorans TG9 were calculated as counts per milliliter (CFU/mL). The viable cells number was measured using a flow cytometer. The 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) (Dojindo, Japan) staining was used to determine the viable cells. Bacteria in the VBNC state still have active respiration. CTC can be reduced by electron transport chains to give off red fluorescence whose intensity reflects the number of viable cells (Zhang et al., 2018 (link)). CTC solution was added to 500 μL of R. biphenylivorans TG9 at a final CTC concentration of 1 mM. Then the suspensions were mixed with 50 μL absolute counting beads (Thermo Scientific, United States). Before analysis, the cell suspensions were incubated at 37°C for 1 h in the dark. When the number of cultured cells had decreased to an undetectable level, the cells still viable were denoted as entering the VBNC state. The VBNC cells number was calculated as the viable cells number minus the culturable cells number (Chen et al., 2019 (link)).
3
Engineered T Cell Proliferation Assay
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Engineered T cells were labelled with CellTrace Violet dye (Thermo Fisher Scientific) and stimulated with MM.1S tumor targets or antigen-coated microbeads at a 1:1 effector:target ratio (non-stimulated control wells were also plated to determine undivided peak for proliferation modeling). After 4 days at 37°C, co-cultures were stained with Live/Dead Fixable stain and antibodies against markers CD8α, CD4, NGFR, CD3, and/or CD138, and mixed with absolute counting beads (Thermo Fisher Scientific). Flow cytometry data were acquired as indicated above, refer to Supplemental Figure 12
4
Characterization of Neoplastic Stem Cells
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Expression of cell surface antigens on primary neoplastic stem cells was analyzed by multicolor flow cytometry using antibodies against CD34, CD38 and CD45 as described [70 (link)]. Putative stem cells were defined as CD34+/CD38−/CD45dim. The gating strategy is shown in Figure A6 . For assessing the absolute numbers of stem cells by flow cytometry, Absolute Counting Beads (Thermo Fisher Scientific, Waltham, MA, USA, order number: C36950) were used according to the instructions by the manufacturer. For the flow cytometric detection of cytoplasmic pSTAT5, cells were first stained for cell surface antigens and then fixed in formaldehyde (2%). Cells were subsequently permeabilized by exposure to 50% methanol (−20 °C, 10 min), washed in phosphate-buffered saline containing 0.1% bovine serum albumin (order number: A4503; Merck) and stained with the Alexa 647-conjugated anti-pSTAT5 mAb 47 pY694 or an isotype-matched control antibody for 30 min at RT. Cells were then washed and analyzed on a FACSCanto (BD Biosciences). Staining reactions were expressed as median fluorescence intensity (MFI). pSTAT5 expression levels are shown as staining index (SI) defined as MFI produced by anti-pSTAT5 antibody:MFI of the isotype-matched control antibody.
5
PBMC isolation and anti-SIRP stimulation
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Peripheral blood mononuclear cells (PBMC) were isolated from Trima residuals of healthy individuals with Ficoll-Paque Plus. 500,000 PBMCs were incubated in U-bottom 96-well plates (Falcon) with anti-SIRP at a concentration of 10 ug/mL for 48 h at 37C.
For quantification of PBMC subsets by flow cytometry, cells were incubated in human FcR blocking reagent and stained with a cocktail of fluorochrome-labeled antibodies against lin—(CD3, CD14, CD16, CD19, CD56) and HLADR. Fixable viability dye was used to identify live cells. After staining, cells were washed and fixed with 0.5% paraformaldehyde in PBS. Prior to acquisition, absolute counting beads (Thermo Fisher) were added and samples were acquired with Canto II flow cytometer and analyzed using FlowJo software.
For quantification of PBMC subsets by flow cytometry, cells were incubated in human FcR blocking reagent and stained with a cocktail of fluorochrome-labeled antibodies against lin—(CD3, CD14, CD16, CD19, CD56) and HLADR. Fixable viability dye was used to identify live cells. After staining, cells were washed and fixed with 0.5% paraformaldehyde in PBS. Prior to acquisition, absolute counting beads (Thermo Fisher) were added and samples were acquired with Canto II flow cytometer and analyzed using FlowJo software.
6
Ex vivo Expansion of Human HSPCs
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Human HSPCs were cultured in serum-free conditions with StemSpan SFEM II (StemCell Technologies, 9655) supplemented with 1% penicillin-streptomycin, 1% l-glutamine, human FLT3-L (100 ng ml -1 ), human TPO (50 ng ml -1 ), human SCF (125 ng ml -1 ) (Thermo Fisher Scientific, 15140122, 25030081, PHC9411, PHC9513 and PHC2113), human low-density lipoprotein (10 μg ml -1 ), SR1 (500 nM) and UM171 (35 nM) (StemCell Technologies, 2698, 72352 and 72914). Cells were cultured at 37 °C and 5% CO 2 , re-plated as necessary to maintain a cell density of ≤1 × 10 6 per ml, and half medium changes were performed every other day. For HSPC expansion assays, cells were analysed by flow cytometry every 5-14 days. Absolute counting beads (Thermo Fisher Scientific, C36950) were used to determine cell numbers and to calculate expansion rates. For co-culture with ECs, E4EC cells were plated in standard medium (M199 with serum and cytokines, see above) the day before adding HSPCs. When co-cultured with HSPCs, the standard supplemented StemSpan medium was used.
7
Chemotaxis Assay for Bone Marrow-Derived Macrophages
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Bone marrow cell suspensions were collected by flushing femurs and tibias of WT C57BL/6 mice (Charles River) and cultured in completed DMEM with 20 ng/mL mouse recombinant M-CSF (catalog no.576406, Biolegend). Fully differentially BMDMs were collected at Day 7 for chemotaxis assay. 10,000 BMDMs in 100 μL/well R10 medium were added onto the top chamber of a 12 well Transwell plate (catalog no.3421, Corning) and 600 μL/well of 20,000 DMBA3-4 or DMSO3-1 cells containing blocking antibody for mouse CSF1R at 20 μg/mL (catalog no.BE0213, BioXCell) or IgG control (catalog no.BE0289, BioXCell) were loaded in the bottom chamber. Migrated cells were collected from the bottom chamber after 96 hours for flow analysis. The average MFI of CD11b and F4/80 were analyzed for CD45+ cells, and the migrated numbers were determined by the ratio of CD45+CD11b+F4/80+ cells to absolute counting beads (catalog no.C36950, Thermo Fisher Scientific).
8
Quantitative M. ovipneumoniae Inoculum Preparation
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M. ovipneumoniae inoculum was grown from a previously described nasal wash isolate (MSU NW-4) and expanded exactly as described in the original publication [11 (link)]. Briefly, inoculum was grown at 37 °C in Mycoplasma broth under microaerophilic conditions. On the day of inoculation, media was removed (10,000× g, 10 min, 4 °C) and culture was resuspended in sterile FACS buffer (2% fetal bovine serum and 0.1% sodium azide in DPBS) for counting by flow cytometry [67 (link)]. Cells were subsequently stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) at 4 °C for 30 min and Absolute Counting Beads (Invitrogen, Carlsbad, CA, USA) were added to solution immediately prior to analysis using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
9
Evaluating ABT-737 Effects on T Cell Subsets
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For in vitro experiments, ABT-737 (AbbVie Bioresearch) was dissolved in DMSO at a concentration of 5 mM and then further diluted in RPMI medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-mercaptoethanol. Either freshly isolated, in vitro IL-2- (R&D Systems, PeproTech) stimulated (3 days with 100 U/ml) or alloantigen-stimulated FoxP3-GFP splenocytes (see iTregs below) were plated in 96-well plates in culture medium at a concentration of 2.5 × 106 cells/ml. Different amounts of ABT-737 or DMSO vehicle control were added to cells and incubated for 12 h at 37°C and 5% CO2. Cells were washed 3×, stained, and viability of CD4+(CD25+)GFP+ and CD4+(CD25−)GFP− cell populations was assessed by PI or 7-AAD exclusion. Absolute cell counts per well were determined by the addition of absolute counting beads (Invitrogen). Surviving cell numbers of each population were normalized to the corresponding vehicle condition.
10
Flow Cytometric Characterization of ILC2s and Th2 Cells
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For the ILC2 analysis, the isolated MNCs were stained with a cocktail of the following antibodies: CD3e (145-2C11, BioLegend), CD11b (M1/70, eBioscience), CD11c (N418, BioLegend), CD45R (RA3-6B2, eBioscience), CD45 (30-F11, BioLegend), ST2 (RMST2-2, eBioscience), KLRG1 (2F1, eBioscience), and CD226 (10E5, BioLegend). ILC2s were characterized as a subpopulation of Lin (CD3e, CD11b, CD11c, and CD45R)− CD45+ cells that expressed KLRG1 and ST2. For intracellular cytokine staining, the MNCs were stimulated with PMA/Ionomycin/BFA for 5 h. After extracellular staining, the cells were fixed, permeabilized, and incubated with anti-IL-5 (TRFK5, eBioscience) and anti-IL-13 (eBioBA, eBioscience) antibodies. For Th2 cell analysis, CD4 (RM4-5, eBioscience), CCR6/CD196 (29-2 L17, BioLegend), and CCR4/CD194 (2G12, BioLegend) antibodies were used. The Th2 cells were defined as CD4+ CCR4+ CCR6− cells. Absolute counting beads (Invitrogen, USA) were employed to calculate the absolute cell number. FMO (Fluorescence Minus One) and matched isotype control were used as controls. The FCM data were obtained using a spectral cell analyzer (SONY SA3800) and analyzed with the Novoexpress software (Agilent Technologies).
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