Anti tnf

For confocal microscopy, Rh-BMDMs were grown and infected in chamber slides (Lab-Tek™ II Chambered cover glass, 2 Well, 4.0 cm2 (Cat#155379, Thermo scientific, Nunc), as described earlier [12] (link). Adherent cells were washed two-times with warm PBS, fixed with 2% paraformaldehyde (Affymetrix) for 1 hour at RT and were either stored at 4°C or directly used for immunostaining. The use of the anti-Mtb antibody (Cat#ab905, 1∶200 dilution) for detection of Mtb is well established and has been used earlier [12] (link)–[16] (link). An antibody against Ln5 (Cat No. 18-0165 Zymed/Invitrogen Inc, 1∶50 dilution) was used to stain Rh-BMDMs and anti-TNF (Cat No. 558882, BD Bioscience, 1∶10 dilution) for detection of TNF.

Immunostaining and confocal microscopy procedures were described previously^{13 (link),27 (link)}. Briefly, RhBMDMs were grown in chamber slides (Lab-Tek), infected with Mtb (MOI, 10:1), and fixed with 2% paraformaldehyde (Affymetrix) for 1 h at room temperature. The use of anti-Mtb antibody (Cat# ab905, Abcam 1:200 dilution) for detection of Mtb, Ln5 (Cat# 18-0165, Zymed/Invitrogen Inc1:50 dilution) for RhBMDMs, and anti-TNF (cat no. 558882, BD Biosciences, 1:10 dilution) for detection of TNF has been described^{11 (link)}. The anti-Mtb antibody and Ln5 were used to mark Mtb and macrophages, respectively^{27 (link),74 (link)}. Cells were also stained for LAMP-1 (Cat# sc-20011, Santa Cruz) and vacuolar proton ATPase, vATPase (Cat# sc-374475, Santa Cruz, 1:200 dilution in in 2% BSA/PBS) and anti-beta actin (Cat# Ab119716) antibodies as described^{38 (link)}. Macrophages treated with both rapamycin (Rap, Cat# SC-3504, Santa Cruz) and bafilomycin A1 or BafA1 alone were stained to measure autophagy levels using anti-LC3B antibody (Cat# L7543, Sigma) as per manufacturer’s instructions. Cells supplemented with recombinant protein TNF-alpha (Cat# 90018-CNAE-5, Thermo Fisher Scientific) as control were also used to detect autophagy levels.

Spleens were removed from mice and single-cell suspensions were prepared by mashing the spleen using a 3-ml syringe plunger on a strainer (70 μM) and washing cells with PBS. Single cell suspensions were stained for flow cytometric analysis with anti-CD3-APC, anti-CD4-Pac Blue, anti-CD8-Pac Orange, Anti- B220-Per CP, anti-annexin-FITC, and anti PI-PE (BD Pharmingen, San Diego, CA). Blood and peritoneal fluid were also harvested and single-cell suspensions were prepared. Cells were stained with Gr-1-FITC, B220-Per CP, CD11b-APC, CD3-Pac Blue, CD11c-PE-Cy7; or CD4-Pac Blue, CD8-Pac Orange, Ly49D-FITC, NK1.1-PE, CD127-APC.
To measure production of cytokines on a per cell basis, splenocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 30 ng/mL) and ionomycin (400 ng/mL) in the presence of 10 μg/mL of Brefeldin A. After 18 hours, cells were surface stained with anti-CD4 and anti-CD8 and processed with an intracellular staining kit (BD Biosciences) according to manufacturer’s instructions. Intracellular antibodies included anti-interferon (IFN)-γ (eBioscience, San Diego, CA), anti-TNF, and anti-IL-2 (both BD Biosciences). Data were acquired on a LSR II multicolor flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).