Mouse primary mixed glial cells were cultured from the cerebral cortices of 1- to 3-day-old mice, as described in our previous study67 (link). The proportion of microglia in murine mixed glial cultures was demonstrated to be 30–50% by FACS analyses using an anti-CD11b antibody (eBioscience, 11-0112, 5 μg ml−1). Neuron-enriched mesencephalic cells were cultured from embryonic day 14 mice as described previously67 (link). In brief, ventral mesencephalic tissues were dissected and incubated in Ca2+-, Mg2+-free HBSS (CMF-HBSS) for 10 min and a 0.01% trypsin in CMF-HBSS for 9min at 37°C. The cultures were rinsed twice in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 6mgml−1 glucose, 204μg ml−1L -glutamine and 100U ml−1 penicillin/streptomycin (P/S) for trypsin inhibition, and then dissociated into single cells by trituration. Cells were seeded onto plates (2 × 106 cells per well) precoated with poly-D -lysine (5 mg ml−1) and laminin (0.2 mg ml−1).
Anti cd11b antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CD11b antibody is a laboratory reagent used for the identification and analysis of CD11b-expressing cells. CD11b is a cell surface marker expressed on various immune cells, including monocytes, macrophages, and neutrophils. This antibody can be used in flow cytometry, immunohistochemistry, and other immunological techniques to detect and characterize CD11b-positive cell populations.
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Lab products found in correlation
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm buffer, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Imdm medium, by Thermo Fisher Scientific (1 mentions)
Rat igg2b, by Thermo Fisher Scientific (1 mentions)
β mercapthoethanol, by Merck Group (1 mentions)
Anti cd11a, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti cd11b antibody
1
Primary Murine Glial and Neuronal Cell Cultures
2
Evaluating Cellular Necrosis in Mtb Infection
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Cellular necrosis was evaluated by staining adherent cells with Fixable Viability Dye eFluor780 (eBioscience), according to the manufacturer’s protocol. Briefly, uninfected and Mtb-infected cells were first stained with Live/Dead staining solution (1:500 diluted in 1× PBS) at room temperature for 10 min in the dark and then incubated with anti-CD11b antibody (eBioscience) for an additional 20 min. Cells were next washed with 1× PBS following centrifugation at 1,500 rpm for 5 min and fixed with cytofix/cytoperm buffer (eBioscience) for 1 h at 4°C. Macrophages were then detached, washed, resuspended in 1× PBS with 1% BSA (MP Biomedicals) and analyzed by flow cytometry.
3
Assessing Mycobacterium tuberculosis-induced Macrophage Necrosis
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Mtb was grown in complete 7H9 broth media at 37 °C for 7 d and bacterial concentration determined by spectrophotometry at 600 nm. Mtb cultures were centrifuged at 5,000 r.p.m. for 10 min, resuspended in OptiMEM and sonicated for 30 s to reduce bacterial clumping. BMDMs were exposed to either H37Rv or H37Rv-RFP at an MOI of 10 for 3–4 h, washed three times with 1× PBS and then cultured in fresh OptiMEM media. L929-conditioned media were added to the cultures to a final concentration of 2.5% on days 1 and 3.
Necrotic cell death was evaluated by staining adherent cells with Fixable Viability Dye eFluor780 (eBioscience) according to manufacturer protocol. BMDMs were stained with Live/Dead staining solution (1:500 diluted in 1× PBS) at room temperature for 10 min in the dark and then incubated with anti-CD11b antibody (eBioscience) for an additional 20 min. Macrophages were washed with 1× PBS following centrifugation at 450 g for 5 min and fixed with cytofix/cytoperm buffer (eBioscence) for 1 h at 4 °C. Fixed cells were then detached, washed, resuspended in 1× PBS with 1% BSA (MP Biomedicals) and analysed by flow cytometry.
Necrotic cell death was evaluated by staining adherent cells with Fixable Viability Dye eFluor780 (eBioscience) according to manufacturer protocol. BMDMs were stained with Live/Dead staining solution (1:500 diluted in 1× PBS) at room temperature for 10 min in the dark and then incubated with anti-CD11b antibody (eBioscience) for an additional 20 min. Macrophages were washed with 1× PBS following centrifugation at 450 g for 5 min and fixed with cytofix/cytoperm buffer (eBioscence) for 1 h at 4 °C. Fixed cells were then detached, washed, resuspended in 1× PBS with 1% BSA (MP Biomedicals) and analysed by flow cytometry.
4
Isolation and Activation of Peritoneal B1a Cells
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Peritoneal B1a cells were obtained through negative magnetic-activated cell separation with a cocktail of antibodies depleting other than B1a cells achieving more than 90% purity in isolation (Miltenyi Biotec B.V., Utrecht, The Netherlands). B1a cells were subsequently counted and plated in IMDM medium (Invitrogen, Eugene, OR, USA) supplemented with 10% fetal calfserum (FCS; Bodinco, Alkmaar, The Netherlands, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 2 mM L-glutamine (all Gibco Invitrogen, Breda, The Netherlands), and betamercapthoethanol (3.57 x 10 -4 M; Millipore, Amsterdam, the Netherlands). Cells were plated in 96-well plate in density of 1x10 6 /mL in 200L of medium and cultured at 37C and 5% CO2 for 48h in presence of 5g of lipopolysaccharide (LPS, isolated from E. coli strain 055:B5, Sigma, St. Louis, MO, USA; LBP from R&D Systems, Abingdon, UK), isotype control (rat IgG2b, eBioscience, San Diego, CA, USA), anti-CD11b antibody (functional grade, eBioscience, San Diego, CA, USA), or anti-CD11a (functional grade, eBioscience, San Diego, CA, USA) in final concentration 10g/mL. After that supernatant was collected and stored at -80C before measurement of IgM antibody by ELISA. Cells were harvested and processed for analysis by flow cytometry and imaging cytometry.
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