Mouse macrophage nucleofector kit

Rab2a depletion in BMMs was carried out using a Mouse Macrophage Nucleofector kit (Lonza) and Amaxa Nucleofector with On-Targetplus SMARTpool siRNAs (Dharmacon) directed against mouse and human Rab2a (L-040851-01-0005 and L-010533-00-0005) or small interfering nontargeting (siNT; J-001810) siRNA, as described previously (22 (link)). Knockdown efficiency was evaluated via densitometry in Western blotting using Bio-Rad ImageLab version 4.1 software on a Chemi-Doc gel imaging system (Bio-Rad), normalizing Rab2a expression to that of β-actin for each sample.
Retroviral supernatants were generated in HEK 293T cells transfected for 48 h h with pCLXSN derivatives and the ecotropic helper plasmid pCL-Eco (Retromax, Imgenex), as described previously (22 (link)). Retroviral supernatants were added to BMMs (2:5 [vol/vol] ratio) for 48 to 60 h prior to paraformaldehyde (PFA) fixation and 24 to 36 h prior to infection.

HEK293T, MC38, and MEF cells were transfected with plasmids using LIPO3000 (catalog L3000015; Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. BMDMs were transfected with plasmids or siRNA using the Mouse Macrophage Nucleofector Kit (Lonza) according to the manufacturer’s instructions. HEK293T cells were transfected with siRNA using INTERFERin (Polyplus) according to the manufacturer’s instructions. Mouse SHP2 or SIRPα knockdown (SHP2 K_D MEFs and SIRPα K_D BMDMs) was performed by lentiviral expression shRNA. The gRNA was used to generate SENP8-KO HEK293T cells. gRNA sequences were cloned into the LentiCRISPR, version 2, according to a standard protocol. gRNA/Cas9 expression plasmids were transfected, and then cells were selected with puromycin (2 μg/ml); the resulting single colonies were picked and expanded. Related sequences are listed in Supplemental Table 1.

LentiGuide plasmids with the correct gRNA insertion were used as templates to amplify hU6 promoter-gRNA fragments in a 50 µl × 8 PCR reaction system using NEB Phusion polymerase. The following primers were used: forward: 5′-ACC​CAG​AGA​GGG​CCT​ATT​TC-3′; reverse: 5′-CTG​TCC​CTG​TAA​TAA​ACC​CG-3′. The gRNA sequences used are negative control: 5′-GAA​CTC​GTT​AGG​CCG​TGA​AG-3′ (Sanjana et al., 2014 (link)); Atg16l1-a: 5′-ACC​GAA​CTG​CAC​AAG​AAG​CG-3′; and Atg16l1-b: 5′-GGG​TCT​GGT​TGG​CTA​CCT​CG-3′. If the intensity and purity of the PCR products were good, then the rest of the amplified DNA was purified using a Qiagen PCR purification kit and eluted with 200 µl double distilled H₂O. 20 µl of 3 M NaAc (pH 5.2) followed by 500 µl ethanol was added to precipitate DNA at −80°C for at least 1 h. After centrifugation at 18,506 g at 4°C for 20 min, the DNA pellet was washed twice with 70% ethanol and allowed to air dry. DNA was then solubilized using 12 µl sterile double distilled H₂O, and the concentration was checked using Nanodrop. The Mouse Macrophage Nucleofector Kit (Lonza) was used to electroporate 3 µg DNA into 2 × 10⁷ freshly isolated bone marrow cells from Cas9-het mice on an Amaxa Nucleofector II machine following the manufacturer’s instructions. Electroporated cells were differentiated in 20% LCM-containing DMEM as above and harvested on day 7 for use.

After 5 days of differentiation, BMMs were collected and 1 × 10⁶ cells electroporated with 2–3 μM ON‐TARGETplus SMARTpool siRNAs (GE Dharmacon) directed against either mouse Arf6 (L‐043217‐01‐0005), mouse Rab8a (L‐040860‐01‐0005), mouse Rab6a/a′ (L‐040858‐01‐0005), mouse Stx6 (L‐059391‐01‐0005), or a non‐targeting (siNT; D‐001810‐10‐20) siRNA, in an Amaxa^TM Nucleofector II using the Mouse Macrophage Nucleofector^® Kit (Lonza). BMMs were immediately diluted in pre‐warmed medium, plated either onto coverslips in a 24‐well plate, or in a 6‐well plate, and incubated for 72 h prior to infection. Protein depletions were evaluated by Western blotting and densitometric analysis using β‐actin levels for normalization. Retroviral transductions of BMMs were performed using derivatives of pCLXSN and the ecotropic helper plasmid pCL‐Eco (Retromax, Imgenex). Retroviral supernatants were generated as follows: HEK 293T cells were seeded in 10 cm tissue culture dishes at 2.5 × 10⁶ in 20 ml medium and transfected after 24 h with a mix of 800 µl DMEM, 8 µg pCL‐Eco, 8 µg pCLXSN derivative, and 48 µl FuGENE^® 6 (Roche) following the manufacturer's protocol and incubated for 48 h prior to collection. Retroviral supernatants filtered through a 0.45 μm filter were added to BMMs (2:5 ratio v/v), and retroviral transduction proceeded for 24 h before BMM infections was performed.

All siRNAs were purchased from Invitrogen (now Thermo Fisher, Waltham, MA, USA). Specific siRNAs against NR1D1 (stealth^TM RNAi; Nr1d1MSS211361 [3_RNAI]) or NR1D2 (stealth^TM RNAi: Nr1d2MSS221355 [3_RNAI]) were used.
Transfection of wild-type BMMCs were performed with Mouse Macrophage Nucleofector Kit (VPA-1009; Lonza, Basel, Switzerland). BMMCs were suspended in Nucleofector Solution to a final concentration of 2 × 10⁶ cells/100μL. The cells were transfected with 500 nM negative control or 250 nM specific siRNA of NR1D1 and NR1D2, respectively, using the Nucleofector II (Amaxa Biosystems, now Lonza) program Y-001. Subsequently, the transferred cells were placed in a 20% FBS medium and cultured in a 24-well plate. After 24 h, the transfected BMMCs were stimulated with anti-mouse IgE antibody (1μg/mL), IL-33 (1 ng/mL), or LPS (1 μg/mL) in the absence or presence of 10 μM SR9009.