Sample Collection & submission guidelines

Required materials

1. Collection tubes: 0.5 – 0.65mL snap-cap centrifuge tubes with these approximate dimensions: ~3.0 cm in height (9/8 inch), 0.9 cm in diameter (3/8 inch).
2. For more examples of acceptable tubes, see the table below:

   Brand	Catalog #	Description
   VWR	87003-290	Microcentrifuge tubes (0.65mL)
   VWR	20170-293	Microcentrifuge tubes (0.65mL)
   Eppendorf	022363611, Fisher Sci. Cat#: 05-402-18	Eppendorf Safe-Lock Tubes™ (0.5 ml)
   Thermo/Matrix	3743-MTX	Screw top micro tubes (0.5 ml)

3. Temperature-controlled centrifuge [plasma or serum]
4. Potter-Elvehjem homogenizer (Teflon pestle and glass mortar) attached to a variable-speed drill a polytron or a tissuemizer [tissue homogenate only]
5. -80°C freezer. If a -80°C freezer is unavailable, the samples should be stored at as low a temperature as possible. If stored at -20°C the freezer should be of a NON frost free type. The stability of samples has not been assessed at temperatures higher than -70°C.
6. Dry ice
7. Various packing supplies as required
8. Styrofoam-insulated corrugated fiberboard outer packaging containers

required volumes

General principles: for samples that need protein extraction, we need 20mg minimum to extra proteins effectively. For samples that do not need protein extraction, we need 200ul diluted sample per well for singleplex test in duplicate, and 50-200ul (depend on panel) for each mulitplex panel. Total amount of sample volume needed is dependent on experiment design and is clarified in the quote’s description.

Important: if you run both singplex and multiplex assays, please alioquote each sample separately in two tubes, as singleplex and multiplex assays are run by different teams and possibly in different times.

Samples that require protein extraction:

• Cells: >20mg, or 100,000 cells, whichever is more
• Tissues: >20mg.
• PBMC's: PBMCs collected with the BD CPT tubes / 2mLs, 20μL needed for each gel.

Samples that do not require protein extraction:

• Lysates and cell culture supernatants: we load 10ug per lane. If the samples’ total protein contents are measured, at least 2x the loading amount per lane is required (e.g. if we are running 5 blots, each with duplicate of each sample, then 5x2x2x10ug=200ug of total protein worth of lysate is required for each sample).
• Plasma and serum: 1-1.5uL of sample per lane, protein concentration always assumed to be ~50mg/mL, need clarification if samples are lipemic
• Blood: we will spin down to serum and plasma at a $20 per sample, required volume refer to Plasma and serum
• Urin, milk and saliva: need to enrich proteins with a kit (Afyon) $50 per sample plus the cost of the kit. Must have enough materials to guarantee >20ug of total proteins per lane.

Samples that are not covered above:

• Consult us before collecting samples.
  

Sample collection procedures

The collection procedure may vary based on your specific sample. Here are some general instructions. DO NOT Parafilm seal your tubes. If necessary please contact your representative for more instructions.

Plasma:

Whole blood samples should be collected by well trained personnel. Use BD Venous Blood Collection Tubes (Cat. # 367861 or equivalent) to collect samples. The samples should be mixed gently immediately upon collection by inverting the tube four or five times to disperse the anticoagulant. To separate the plasma from the cells, centrifuge the collection tube for 10 minutes at 1,300 x g (or according to the manufacturer's instructions) using a temperature setting of 25 °C. It is best to centrifuge within 30 minutes of sample collection, at most do not exceed 2 hours. The plasma should be aspirated and contained in microcentrifuge plastic tubes. Do not collect the buffy coat that is at the interface between the red cells and the plasma.

Serum:

Samples should be collected in plastic BD Vacutainer® SST™ collection tubes (Cat. # 367988 or equivalent). Following collection, the samples should be allowed to clot in the collection tubes for a minimum of 30 minutes at room temperature. Serum should be separated from the clot by centrifuging the collection tube for 10 minutes at 1,300 xg (or according to the manufacturer’s instructions) using a temperature setting of 25°C, within two hours of collection. If a fixed angle centrifuge is used the time should be increased to 15 minutes. The serum should be aspirated by pipette and stored in plastic microcentrifuge tubes.

Urine:

Urine samples, at least 25 mL, should be collected into a sterile, screw-capped polypropylene container. If a pour-off tube is used, we recommend using a 2 mL screw top polypropylene tube.

Tissue Homogenate:

Tissue samples should be collected, weighed, and added to lysis buffer (100 mg of tissue per 900 μL lysis buffer). Our recommended lysis buffer is 50mM Tris-HCl with 2mM EDTA, pH 7.4. If the samples are not homogenized immediately then the samples should be frozen in liquid nitrogen and stored at -80°C. While EDTA is a good inhibitor of divalent metal requiring proteases, you may want to minimize other protease activity by adding the following inhibitors: aprotinin, antipain, leupeptin, and pepstatin A (all at 1μg/mL) and 2mM PMSF (phenylmethylsulfonyl flouride).

Tissues may be homogenized using a Potter-Elvehjem homogenizer (Teflon pestle and glass mortar) attached to a variable-speed drill, a polytron or a tissuemizer. During the homogenization process, the tube should be submersed in an ice bath to maintain the sample at 2-8 °C. Following homogenization, the tissue preparation is centrifuged for 2 minutes in a microfuge at 13,000 xg. Making sure that the cell pellet is not disturbed; aspirate the supernatant.

Sample Labeling instructions

Please Maintain your own record of which number corresponds to which sample.

Label print and writing must be clear: you can use printed labels on the side of tubes ONLY if you are sure that the label will not fall off in -80°C and remain attached. If labels fall off the tubes, it will result in complete samples lost. If you are hand-writing the labels, please use a felt-tip permanent marker. Make sure the labels are easy to read.

What to print/write on labels: label the tube lids/caps alpha numerically with the first letter of your last name followed by consecutive numbers. For example, if your last name is “Sam” and you are sending three samples your tubes will be labeled: S1, S2 and S3 etc.

Please DO NOT use parafilm to seal your tubes.

packaging: document to include with the shipment

Documents: sample manifest template: Sample Manifest

Shipping information

For singplex samples, ship with dry ice on Monday or Tuesday via

priority overnight or equivalent to:

Attn: Boster Bio C/O Christine Lai

Project Number: [project #, format BOSxxxxx]

3942 Valley Ave, Ste B

Pleasanton, CA 94566

Important Tips

Some common mistakes occur during sample collection, handing and storage are listed below. Please be mindful of them and double check before start collecting samples, and check again before sending out your package.

Avoid sample volume underfill: check each tube visually for obvious underfill. The assays will require sufficient volume to run and a underfilled sample may lead to the compromise of the entire sample, and subsequently the integrity of the entire study.

Keep sample cold: All samples must be frozen immediately after collection. Have a bucket of dry ice ready and put samples on dry ice before they are put in -80°C freezer. If sample has to be stored at higher than -80°C it should be as short as possible. If the samples are stored at -20°C, the freezer must be of a NON frost-free type, as frost-free freezers cycles temperature to above freezing to get rid of frost which will destroy the samples through multiple cycles of freeze-thaw.

Fill out the form before shipping: Boster Bio’s Sample Submission Document must be printed and included with the package. Template: https://docs.google.com/document/d/1HgrGMmrVEGEs86uNUi1Rpa9p0SVsD6PT/edit?rtpof=true&sd=true

Shipping overnight on Mon-Wed: ship the samples to Boster Bio’s facility using overnight shipping. Ship only on Monday through Tuesday, so we can receive the sample Tuesday through Wednesday to leave enough time for receiving and handling.

Use a grid to organize samples: when possible, put samples into a grid instead of a plastic bag. It can be a cardboard box grid or a plastic rack.

Avoid brokage, leaking, thawing: Check the tubes for cap tightness one more time. If the samples show signs of brokage, leaking or thawing, the biological molecules in them will likely have degraded and the data obtained from them will no longer be valid so they might be rejected for analysis. Please do a thorough check and eliminate any risk that could lead to sample compromises.

Required materials

1. hydrophobic markers/pencils (for labeling slides).
2. fixiative (4% paraformaldehide, Boster Bio AR1068, or 10% formalin). Volume of fixiative should be 20x or more than the sample volume, and the tissue should be completely submerged in the fixiative.
3. proper containers such as falcon tubes or histology containers (for fixed tissues).
4. vacum bags (for FFPE and OCT blocks).
5. 70% ethanol.

Sample volumes

General principles: for making FFPE and OCT blocks, the maximum size of tissue per block is 30mm x 24mm x 4mm. During autopsy please isolate region of interest (ROI) only. There is no hard requirement of sample volumes although for cells it is best to provide > 100,000 cells.

Sample collection procedures

We can accept fixed tissues, blocks and slides.

Slides:

Labeling:

Make sure all slides are labeled clearly and include all relevant information.

Slide Placement:

Position the slides carefully in the slide carrier, ensuring they are placed flat and securely.

Container:

Use a piece of tape to securely fasten the lid of the container, ensuring it is tightly sealed.

Packaging:

Place the sealed slide(s) inside a padded envelope or box for protection during shipping.

Additional Protection:

For added security, wrap the container with bubble wrap before placing it in the envelope or box.

Securing:

Use tape to secure the envelope or box, ensuring it is tightly sealed.

Bulk Shipping:

If shipping a large collection of envelopes, consider using a sturdy carton or crate to house the envelopes for added protection.

Tissues:

Labeling:

Ensure that tubes or cassettes containing the tissue samples are properly labeled using a pencil or laboratory-grade markers/labels that are resistant to formalin, ethanol, isopropanol, and xylene.

Make sure the labels are legible and can withstand the subsequent processing steps.

Isolation:

During necropsy or tissue collection, isolate only the region of interest for each tissue sample.

The maximum size of the tissue should not exceed 24mm width × 30mm length × 4mm depth.

Fixative Volume:

The volume of fixative used should be 20 times the volume of the tissue sample.

Ensure that the tissue is completely immersed in the fixative solution to facilitate proper fixation.

Fixative Solution:

You can order pre-filled histology containers with 10% formalin from WR (mention the source/company), or you can submerge each wet tissue specimen in a 50mL Falcon tube filled with 10% formalin.

Seal the cap tightly to prevent leakage.

Submitting Samples:

If you have samples that cannot be immediately analyzed, you can submit them as "process and embed only" for the requested service when placing an online order.

Provide clear instructions regarding the desired service for those specific samples.

Please adhere to these guidelines to ensure proper tissue preparation for subsequent immunohistochemistry analysis.

packaging: document to include with the shipment

Documents: sample manifest template: Sample Manifest

Shipping information

Shipping condition depends on sample type:

Fixed samples and slides can be shipped in room temperature.

Fresh samples and OCT blocks should be shipped with dry ice.

FFPE blocks should be shipped with ice packs to prevent melting.

Ship to:

Attn: Boster Bio CJ Xia

Project Number: [project #, format BOSxxxxx]

3942 Valley Ave Suite B

Pleasanton, CA 94566

Important Tips

Proper labeling: Ensure that all samples and containers are clearly labeled with relevant information such as patient ID, sample type, and collection date. Use permanent markers or labels resistant to common fixatives.

Adequate fixation: Fix tissues promptly in a suitable fixative, such as 10% formalin, to preserve cellular structures. Follow recommended fixation times depending on the tissue type and size. Contact us for more details.

Optimal sample size: When collecting tissues, aim for a size that allows thorough fixation and processing. The recommended maximum size is typically around 24mm width × 30mm length × 4mm depth.

Immersion in fixative: Completely immerse the tissue in the fixative solution, ensuring that the sample is fully covered. The volume of fixative should be around 20 times the volume of the tissue.

Proper packaging: Use leak-proof containers or tubes to hold the fixed tissues. Consider using pre-filled histology containers with the appropriate fixative. Seal the containers tightly to prevent any leakage during shipping.

Secondary packaging: Place the sealed containers inside a padded envelope or box for added protection. If desired, wrap the containers with bubble wrap or other cushioning material to prevent damage during transit.

Secure packaging: Use tape to secure the envelope or box, ensuring that it is properly sealed. If shipping a large collection of samples, consider using a sturdy carton or crate to house the envelopes for added stability.

Documentation: Include all necessary documentation, such as requisition forms and shipping manifests, to accompany the samples. Keep copies of these documents for your records.

Communication: Clearly communicate with Boster Bio regarding the contents of the shipment, any special handling instructions, and anticipated delivery dates.

Shipping information

cDNA can ship in room temperature. Tissues and cells should be flash frozen and ship on dry ice.

Ship on Monday to:

Attn: Boster Bio CJ Xia

Project Number: [project #, format BOSxxxxx]

3942 Valley Ave Suite B

Pleasanton, CA 94566

Important Tips

Sample preservation: Depending on the nature of the samples, use suitable preservation methods to maintain sample integrity. This may include snap-freezing samples, storing them in RNA stabilization reagents, or keeping them at specific temperatures.

Sample labeling: Clearly label each sample tube or container with relevant information, such as sample ID, date, and any specific requirements. Use permanent markers or labels that are resistant to common storage conditions and sample treatments.

Packaging materials: Choose appropriate packaging materials that provide insulation and protection during shipping. Use leak-proof, sterile, and non-reactive containers or tubes that can withstand temperature fluctuations and handling during transit.

Documentation: Prepare necessary documentation, including sample manifests, shipping labels, and any required customs forms. Include relevant information about the samples, such as sample type, quantity, and storage conditions.

Required materials

1. Cell seeding plates, sealer cap mats, and shipping vacuum bags (can be purchased from Boster Bio, usually included in the project quote). Or use plates equivalent to Greiner Bio-One plates: #6545097 (Clear bottom tissue culture plates with black walls), with capping sealer that can sufficiently prevent leaking.
2. Heat vacuum sealer. The common ones for the kitchen are sufficient. Costco sells it for $75 as of 2023.

required volumes

Desired density is 25,000 cells per well.

Sample collection procedures

Seed, treat and fix cells prior to shipping overnight on dry ice.

The experiment typically constitutes 2 phases. We need to run the assay on the day that the plate arrives. Send fresh samples for each phase immediately before the experiment to avoid storing the cells for extended periods of time. Please contact us to coordinate your cell treatments and shipments.

For the optimization phase, we will decide the optimal cell seeding density and antibody concentration to use. Please seed your plate according to the following instructions: 

1. In the 96-well plate, for each 12-well row, seed, from the top (row A) to bottom (row H), 

A: 100,000 cells/well

B: 50,000 cells/well

C: 25,000 cells/well

D: 12,500 cells/well

E: 6,250 cells/well

F: 3,125 cells/well

G: 1,563 cells/well

H: 0 cells/well

2. Discard media and apply 50μL of 100% Methanol to each well and incubate at -20°C for 20 minutes.

3. Discard Methanol and apply 300μL of 1X PBS

4. Place a cap mat on the plate and vacuum seal in the provided bags.

And label this plate clearly with “For optimization”. Indicate this in the sample manifest as well.

Once the optimal seeding density has been decided, in the second phase: 

1. Seed and treat cells as desired at a density decided in previous phase and incubate overnight at 37°C, 5%CO2.

2. Discard media and apply 50μL of 100% Methanol to each well and incubate at -20°C for 20 minutes.

3. Discard Methanol and apply 300μL of 1X PBS

4. Place a cap mat on the plate and vacuum seal in the provided bags.

*our lab has done stability testing on the in-cell western plates and found them stable if left in methanol at -20°C for up to a year.

Sample Labeling instructions

A plate layout file must be provided to illustrate which wells contain which samples, and what measurement should be done to each well. Each plate should be labeled clearly with cold-resistant markers. Use the sample manifest template below is preferred.

packaging: document to include with the shipment

Documents: sample manifest template: Sample Manifest

Shipping information

First contact us to make sure we have availability to process your samples upon arrival. Once confirmed,

Ship with ice on Monday or Tuesday via

priority overnight or equivalent to:

Attn: Boster Bio C/O Christine Lai

Project Number: [project #, format BOSxxxxx]

3942 Valley Ave, Ste B

Pleasanton, CA 94566

Required materials and information

1. Packaging ready vector(s) containing GOI(s).
2. Vector map and full sequence is required for each vector. Please also indicate if any reporter expression is expected. The expected reporter expression, e.g. GFP, should not require induction of expression.
3. The amount and estimated concentration of each vector is required.

required volumes

If gene amplification service is ordered, send send >10ug of each vector at >0.5μg/μl.

If gene amplification service is not ordered, send the minimum required amount of DNA for each project. See the AAV packaging service page for minimum DNA required for each titer production.

If gene synthesis service is ordered, no material is required.

Sample Labeling instructions

Each vector's container/tube should be clearly labeled with water permenant markers so it can be matched to the vector maps information.

packaging: document to include with the shipment

Documents: sample manifest template: Sample Manifest

Shipping information

Ship with ice on Monday or Tuesday via

priority overnight or equivalent to:

Attn: Boster Bio C/O CJ Xia

Project Number: [project #, format BOSxxxxx]

3942 Valley Ave Suite B

Pleasanton, CA 94566