COPARC Biorepository Methods 6-6-12

Microbiome Oral Wash Processing (Alcoholics and Controls)

Notes:

§  Decontaminate all surfaces with a 10% bleach solution or other DNA denaturing product.

§  Prepare all reagents, labels, vials, etc. prior to procedures in a laminar flow hood.

§  All processing must be in a laminar flow hood under DNA-free conditions.

§  Collect, process, and run saline controls in parallel to clinical samples.

§  Oral wash should be kept on ice until processing. Processing should be done as soon as possible, but absolutely within 20 minutes.

  1. Transfer the oral wash and saline control to sterile 15mL conical tubes and centrifuge at 4°C, 5000 x g for 20 minutes.
  2. Discard the supernatant and resuspend the pellet in 0.5mL sterile PBS.
  3. Transfer to a labeled 1.8mL cryotube (Nunc #375418) with a grey cap (Nunc #375906).
  4. Store at -80°C.

Microbiome Bronchial Brushings Processing

(Alcoholics and Controls)

Notes:

§  Decontaminate all surfaces with a 10% bleach solution or other DNA denaturing product.

§  Prepare all reagents, labels, vials, etc. prior to procedures in a laminar flow hood.

§  All processing must be in a laminar flow hood under DNA-free conditions.

§  Collect, process, and run saline controls in parallel to clinical samples.

§  The brushes and saline controls will arrive in the lab in sterile cryotubes with grey caps and will have been vortexed once and placed on ice.

  1. Vortex on highest setting for 1 minute
  2. Remove brush in the laminar flow hood using DNA-decontaminated forceps.
  3. Store at -80°C.

Non-microbiome Bronchial Brushings Processing

(Alcoholics and Controls)

Note: The brush will arrive in the lab in a sterile cryotube with a grey cap containing RNAlater (Qiagen #76106) and has been vortexed once and placed on ice.

1.  Store the cryotube at 4°C for at least 48 hours to allow the RNAlater to penetrate the brush. DO NOT freeze immediately.

2.  Store the cryotube at -20°C until shipment. Transport for analysis within 2-4 months at ambient temperature, except when the temperature is >90°F, when wet ice should be used.

Exhaled Breath Condensate Processing (All Patients)

Note: The RTube (Respiratory Research, Inc #2501) should be capped and stored on ice until ready to process. It should be processed within 20 minutes after collection.

  1. Gently roll the RTube to collect all of the sample off the side of the tube.
  2. Remove the caps from both sides of the RTube and pool the sample by using the RTube plunger. Ensure the red up arrow is properly oriented while pushing the RTube down onto the plunger.
  3. Aliquot 110μl of samples into Nunc 1.8mL cryotubes with blue cryo caps (Nunc #354879).
  4. Store at -80°C.

Exhaled Breath Condensate for HPLC

Notes:

§  The RTube (Respiratory Research, Inc #2501) should be capped and stored on ice until ready to process. If study staff is unable to process sample immediately, R-tubes can be capped and frozen in -80°C freezer immediately after collection and should be processed later the same day.

§  The Brown Lab at Emory University will supply the color coded tubes and the preservation solutions. The tubes with preservation solution should be stored at -20°C and thawed on ice before use.

§  Ship specimens on dry ice once every two months or more frequently if desired to Frank Harris at the Brown Lab. Send an email or call first to make sure someone is there to receive the samples.

  1. Gently roll the RTube to collect all of the sample off the side of the tube.
  2. Remove caps from both sides of the RTube and pool the sample by using the RTube plunger. Ensure the red up arrow is properly oriented while pushing the RTube down onto the plunger.
  3. Aliquot 200μl into the blue tube (glutathione HPLC) and immediately place on ice.
  4. Aliquot 50μl into the pink tube (urea) and immediately place on ice.
  5. (Optional) Aliquot 250μl into the orange tube (isoprostane) and immediately place on ice.
  6. (Optional) Aliquot the remaining EBC into the brown tube and immediately place on ice.
  7. Vortex all samples to mix well.
  8. Store samples at -80°C for no longer than 2 months.

Blood Processing (All Patients)

Note: For blood draws use at least a 21G needle to minimize hemolysis. Also avoid aggressive suction during phlebotomy for this same reason.

Blood draw for serum, plasma, and buffy coat collection

Centrifuge tubes at 900X g for 10 min at room temperature.

Green top (sodium heparin)(BD Vacutainer 4mL #367871, 6mL #367878) - aliquot 0.5mL aliquots of plasma off the top phase into 1.8mL cryotubes (Nunc #375418) with green cap inserts (Nunc #355018), then collect red blood cells (RBCs) from the bottom phase and aliquot them into 1.8mL cryotubes with yellow cap inserts (Nunc #355077). A 4mL green top will typically yield between 1 and 3mL of plasma.

Red top (BD Vacutainer 4ml #367812, 6mL #367815, 10mL #367820) – aliquot 0.5mL aliquots of serum off of the top phase into 1.8mL cryotubes with red cap inserts (Nunc #354968). A 6mL red top will typically yield between 1 and 4mL of serum.

Purple top (EDTA)(BD Vacutainer 4mL #367861, 6mL #367863) – aliquot 0.5mL aliquots of plasma off the top phase into 1.8mL cryotubes with purple cap inserts (Nunc #375922). A 4mL purple top will typically yield between 1 and 3mL of plasma. Then collect the buffy coat by pipetting the white layer of cells between the top phase and the red blood cell (RBC) bottom layer and pipetting it into a 1.8mL cryotube with a pink cap insert (Nunc #375884).

Light Blue Top (Na Citrate) (BD Vacutainer 4.5mL #363083) – aliquot 100μl aliquots into 1.8mL cryotubes with white cap inserts (Nunc #354755).

Optional: Add 10uL of 100uM BHT (from McCord Lab) per mL of blood to EDTA plasma, sodium heparin, plasma, and serum for TBARS and Pon1 assays.

Blood draw for HPLC measurements

1.  Draw blood into syringe and immediately add 1 mL of whole blood to 500uL of blood preservation solution (from Brown lab) in a clear 1.5mL microcentrifuge tube (Fisher Scientific #05-408-129). Note: Use at least a 21G needle to minimize hemolysis.

  1. Centrifuge for 10 min at 500 X g
  2. Aliquot 250uL of the upper phase into a green 1.5mL microcentrifuge tube (Fisher Scientific #05-408-133) containing 250uL GSH preservation solution (from Brown lab).
  3. Store at -80°C.

BAL Processing (Microbiome – Alcoholics and Controls)

Notes:

§  Decontaminate all surfaces with a 10% bleach solution or other DNA denaturing product.

§  Prepare all reagents, labels, vials, etc. prior to procedures in a laminar flow hood.

§  All processing must be in a laminar flow hood under DNA-free conditions.

1.  Prior to centrifugation for isolation of alveolar cells (see below), remove 2mL of ‘Lower’ BAL and place into a clear 2mL microcentrifuge tube (Fisher# 05-408-138). Centrifuge at 25 X g for 1 minute to settle the cells.

2.  Without disturbing the cell pellet, very gently pipette out 1.5mL of the cell-free lavage and transfer to a labeled clear 2mL microcentrifuge tube.

3.  Centrifuge at 5000 X g for 20 minutes. Decant off supernatant.

4.  Store the pellet at -80°C.

BAL Processing (Alcoholics and Controls)

Notes:

§  In general, 150-200mL sterile isotonic saline will be used for bronchoalveolar lavage, instilled in 50mL aliquots. We have used hand aspiration to more carefully control suction amount in the lung.

§  The first 50mL aliquot instilled/aspirated is reflective of the airways, and is kept separate from the subsequent aliquots and should be marked ‘Upper’. The volume aspirated from this first aliquot (assuming 50mL instilled) is generally only 10-20mL.

§  The second, third, and any subsequent aliquots reflect distal airway/alveolar sampling and should be marked ‘Lower’.

§  Aliquot cell free BAL in a biosafety cabinet (hood).

1.  Record volume received.

2.  Centrifuge the 50mL conical tubes of BAL fluid 900 x g for 10 min at room temperature.

3.  Place samples on ice while aliquotting.

BAL for HPLC Measurements: For ‘Upper’ and ‘Lower’ add 500uL BAL and 100uL GSH preservation solution (from Brown lab) to a yellow 1.5mL microcentrifuge tube (Fisher Scientific #05-408-131), add 500uL BAL and 500uL GSH preservation solution to a blue 1.5mL microcentrifuge tube (Fisher Scientific #05-408-132), and add 1.5mL BAL to an amber 1.5mL microcentrifuge tube (Fisher Scientific #05-408-134). There should be one set of tubes for the ‘Upper’ samples and one set of tubes for the ‘Lower’ samples.

Optional: For ‘Lower’ BAL, add 10uL of 100uM BHT (from McCord lab) per mL of BAL to 4mLs of BAL and aliquot into 1.8mL cryotubes (Nunc #375418) with orange cap inserts (Nunc# 355158) for TBARS and Pon1 assays.

4.  Aliquot remaining BAL in 3-3.5mL aliquots in sterile 5mL screw top tubes (autoclaved Sarstedt #60.612.012 and #65.729 or Nunc #348100).

5.  Store all BAL samples at -80°C.

BAL Cell Processing

  1. In a biosafety cabinet (hood), resuspend the cells from the above aliquots (‘Upper’ and ‘Lower’) in a total of 10mL 10mL RPMI (Cellgro #10-040-CV) and antibiotics (Cellgro #MT-30-002-CI). Place 2-3mL of media in each 50mL conical tube, gently resuspend the cells, and then combine all of the resuspended cells into one tube.
  2. Count cells and check viability by diluting 10μl of sample with 90μl trypan blue (VWR #45000-717) and applying 10μl of the dilution to a hemacytometer. Count all of the cells in the 4 quadrants then divide that number by 4. Multiply that number by 104 to calculate how many cells there are per mL. Multiply that number by the total volume (10mL) to calculate the total number of cells in the 10mL of cell suspension. An automated cell counter can also be used, if available.

3.  Divide up the cells according to assay need below.

Cytospins: Resuspend 120,000 macrophages in 400ul of 1XDPBS (Cellgro #21-031-CV). Cytospin 4 slides (Fisher Scientific #12-544-7) at 30,000 cells per slide, following the equipment’s instructions. Immediately after cytospin, fix 2 of the slides in 2% paraformaldehyde (Electron Microscopy Sciences #15710, diluted in 1XDPBS) for 15 minutes at room temperature. Immediately place slides in 70% EtOH for 1 minute, remove and store in 1XDPBS at 4°C. The remaining 2 slides are stained using Protocol Hema 3 (Fisher Scientific #22-122-911), dried, and stored in a slide box at room temperature for subsequent tabulation of differentials.

Cells for RNA: 1- 2x10 6 of cells are centrifuged at 900 X g for 5 minutes to pellet the cells. The media is aspirated off and discarded, and then the cells are immediately frozen and stored at -80°C.

Cells for proteins: 2-4x10 6 of cells are centrifuged at 900 X g for 5 minutes to pellet the cells. The media is aspirated off and discarded, then the cells immediately frozen and stored at -80°C.

Freeze down protocol for cryostorage (For BAL cells and PBMCs): Pellet cells by centrifuging them at 900 X g for 5 minutes. Resuspend them with room temperature DMEM freezing media (35mL DMEM-Cellgro #10-017-CV, 10mL FBS-Atlanta Biologicals #S11550H , 5mL DMSO- Sigma #D2650, filter sterilized -Corning #430320) at a final concentration of 2-4 x106, aliquot into 1.8mL cryotubes (Nunc #375418), and place on ice. Place them in a freezing container (Nalgene Cryo 1°C Freezing Container or Biocision Cool Cell) and store at -80°C for 24 hours. Store the cells in a liquid nitrogen storage tank long term.

Secretion of cytokines/growth factors by treated cultured Alveolar Macrophages (AMs) in media:

  1. Plate 500,000 macrophages in 2mL RPMI (Cellgro #10-040-CV) and antibiotics (Cellgro #MT-30-002-CI) per well in a 12 well plate (Costar #3513), incubate at 5% CO2 and 37°C for at least 1 hour to allow the cells to adhere.
  2. Dilute 1mg/mL stock LPS (Sigma #L-6529) in 10mL RPMI with antibiotics to a concentration of 2ug/mL.
  3. Dilute 1mg/mL stock LTA (InvivoGen #tlrl-pslta) in RPMI with antibiotics to a concentration of 10ug/mL.
  4. Dilute 1M NAC (Sigma #A9165) in RPMI with antibiotics to 5mM.
  5. AMs only: For two of the macrophage plated wells, aspirate the media and replace with 2 mL of fresh RPMI with antibiotics.
  6. AMs + LPS: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and LPS and 1mL of RPMI with antibiotics for a final LPS concentration of 1ug/mL.
  7. AMs + LTA: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and LTA and 1mL of RPMI with antibiotics for a final LPS concentration of 5ug/mL.
  8. AMs + NAC: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and NAC and 1mL of RPMI with antibiotics for a final NAC concentration of 2.5mM.
  9. AMs + LPS + NAC: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and NAC and 1mL of RPMI with antibiotics and LPS.
  10. AMs + LTA + NAC: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and NAC and 1mL of RPMI with antibiotics and LTA.
  11. Incubate the plate O/N at 5% CO2 and 37°C.
  12. The next day, collect the media in 1mL aliquots, 2 aliquots per well and store at -80°C. Well 1 aliquots should be labeled 1A and 1B and well 2 aliquots should be labeled 2A and 2B.
  13. Save the cells for RNA extraction by adding 350μl Buffer RLT from an RNeasy Mini Kit (Qiagen #74104) with β-mercaptoethanol (Sigma #63689) to each well in order to lyse the cells. Scrape the cells, collect all of the Buffer RLT and cells from each condition and load the collected sample onto a QiaShredder column (Qiagen #79654) placed inside a 2mL collection tube from the kit. Centrifuge at max speed for 2 minutes. Discard Qiashredder column and cap the tube with sample with the provided caps and store the homogenized sample at -80°C for up to 3 months.

BAL and BAL Cell Processing (Burn Patients)

Notes:

§  BAL will be immediately transported on ice to the lab.

§  Aliquot BAL in a biosafety cabinet (hood).

§  Wash buffer – PBS(Gibco #14190) with 5% FBS (Gibco #16000, certified US origin) and 1% PSG(Gibco #10378)

§  Media - RPMI (Gibco #21870) with 5% FBS (Gibco #16000, certified US origin) and 1% PSG(Gibco #10378)